Uronic Acids in Oligosaccharide 1 1

13-2-2007 16:49:03

      Uronic Acids in Oligosaccharide Synthesis                PROEFSCHRIFT        ter verkrijging van  de graad van Doctor aan de Universiteit Leiden,  op gezag van Rector Magnificus prof. mr. P.F. van der Heijden,  volgens besluit van het College voor Promoties  te verdedigen op woensdag 18 april 2007  klokke 13.45 uur      door        Leendert Johannes van den Bos  geboren te Bruinisse in 1979 

1

Promotiecommissie    Promotores 

: 

Prof. dr. G.A. van der Marel 

 

 

Prof. dr. H.S. Overkleeft 

:  

Prof. dr. C.A.A. van Boeckel (NV Organon) 

Overige leden 

: 

Dr. G. Lodder 

 

 

Dr. J.D.C. Codée 

 

 

Prof. dr. J. Lugtenburg 

 

 

Prof. dr. J. Brouwer 

 

 

Prof. dr. J.P. Kamerling (Universiteit Utrecht) 

  Referent   

Coverdesign:  Van der Meer Reclameatelier, Bruinisse; www.vanderM3er.nl.    Gedrukt bij Haveka BV, Alblasserdam.  De totstandkoming van dit proefschrift werd mede mogelijk gemaakt door de Nederlandse  Organisatie voor Wetenschappelijk Onderzoek en de J.E. Jurriaanse Stichting. 

2

 

 

 

3

      Table of Contents   

4

List of Abbreviations   

6 

Chapter 1  General Introduction   

9 

Chapter 2  Thioglycuronides: Synthesis and Application in the Assembly of Acidic  Oligosaccharides   

31 

Chapter 3  Preparation of 1‐Thio Uronic Acid Lactones and Their Use in Oligosaccharide  Synthesis   

51 

Chapter 4  Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters: Synthesis of an   Alginate Trisaccharide   

65 

Chapter 5  Studies of the Glycosidation Properties of Deactivated   1‐Thio Mannosazidopyranosides               

83 

 

                    Chapter 6  A Novel Strategy Towards the Synthesis of Orthogonally Functionalized   4‐Aminoglycosides   

99 

Chapter 7  Synthesis of a Protected Trisaccharide Repeating Unit of the Zwitterionic  Polysaccharide Sp1   

111 

Chapter 8  Synthetic Study Towards the PSA1 Tetrasaccharide Repeating Unit    

123 

Chapter 9  Summary, Mechanistic Aspects, and Perspective   

141   

Samenvatting   

151 

List of Publications   

153 

Curriculum Vitae   

155 

Nawoord 

157 

 

       

 

5

      List of Abbreviations    Ac   ACN 

acetyl  acetonitrile   

DMTST   

dimethyl(thiomethyl)sulfonium  triflate 

All   Arom   aq.   BAIB   Bn  

allyl  aromatic  aqueous  [bis(acetoxy)iodo]benzene  benzyl 

DTBMP   equiv.   ESI   Et   FAB 

2,6‐di‐tert‐butyl‐4‐methylpyridine  molar equivalents  electrospray ionization  ethyl  fast atom bombardment 

Boc  bs   BSP   Bz   CAA 

tert‐butyloxycarbonyl  broad singlet  1‐benzenesulfinyl piperidine  benzoyl  circulating anodic antigen 

Fmoc   fur.  Fuc  GAG  Gal 

9H‐fluoren‐9‐ylmethoxycarbonyl  furanoside  D‐fucose  glycosaminoglycan  D‐galactose 

cat.   Cbz  cf  ClAc  COSY 

catalytic  benzyloxycarbonyl  confer  chloroacetyl  correlation spectroscopy 

GalA  GalN  Glc  GlcA  GlcN 

D‐galacturonic acid 

Cq   d   DBU   DCE  

quarternary carbon atom  doublet  1,8‐diazabicyclo[5.4.0]undec‐7‐ene  dichloroethane 

GulA  HPLC    HRMS  

L‐guluronic acid  high performance liquid  chromatography  high resolution mass spectrometry 

DCM   dichloromethane  dd   doublet of doublets  DIBAL‐H  di‐iso‐butylaluminium hydride  DiPEA   N,N‐di‐iso‐propyl‐N‐ethylamine  DMAP   4‐dimethylaminopyridine 

Hz   IBX  IDCP     IR  

Hertz  o‐iodoxybenzoic acid  iodonium di‐sym‐collidine  perchlorate  infrared spectroscopy 

DMF   DMSO        

isoprop  J        

isopropylidene  coupling constant 

 

6

N,N‐dimethylformamide  dimethylsulfoxide 

    

D‐galactosamine  D‐glucose  D‐glucuronic acid  D‐glucosamine 

 

   

 

         

         

       

       

    LCMS    Lev 

liquid chromatography mass  spectrometry   levulinoyl 

    q  RP  rT  

quartet  reverse phase  room temperature 

m   M   Man 

multiplet  molar   D‐mannose  

ManA  MBz  MP   Me  

D‐mannuronic acid 

4‐methoxybenzoyl  melting point  methyl 

s   Su  tBu  t   TBABr 

singlet  succinimide  tert‐butyl  triplet  tetra‐n‐butylammonium bromide 

Ms  MS3Å  MS  NIS   NMR  

mesyl   molecular sieves 3 ångström  mass spectrometry  N‐iodosuccinimide  nuclear magnetic resonance 

TBAF   TBAI  TBDPS   TBDMS  TBS  

tetra‐n‐butylammonium fluoride  tetra‐n‐butylammonium iodide  tert‐butyldiphenylsilyl  tert‐butyldimethylsilyl  tert‐butyldimethylsilyl 

ORTEP  p   P  PCC  PDC 

Oakrich Thermal Ellipsiod Plot  para  protective group  pyridinium chlorochromate  pyridinium dichromate 

TCA  TEMPO     TEP   tert  

trichloroacetyl  2,2,6,6‐tetramethyl‐1‐ piperidinyloxy, free radical  triethyl phosphite  tertiary 

PE  Pent  Ph   Phth   pMB 

petroleum ether  pentenyl  phenyl  phthaloyl  p‐methoxybenzyl 

Tf   TFA  THF   TLC  

trifluoromethanesulfonyl  trifluoroacetyl  tetrahydrofuran  thin layer chromatography 

pMP   ppm   Pr  pyr.    

p‐methoxyphenyl   parts per million  propyl  pyridine 

Tol  TMS   Tr  Ts  TTBP  

p‐toluyl  trimethylsilyl  trityl  p‐toluenesulfonyl  2,4,6‐tri‐tert‐butylpyrimidine 

UV  wrt 

ultra violet  with respect to 

     

 

7

8

 

   

Chapter 1 │  General Introduction       

Introduction Uronic acids are an important class of monosaccharides and are defined as aldohexoses that have their primary alcohol oxidized to a carboxylic acid.1,2 Polysaccharides containing uronic acid entities are widely spread in Nature and display an array of physical properties and biological functions. A well known class of polysaccharides are the glycosaminoglycans (GAGs), which are composed of uronic acids linked to 2-acetamido-2-deoxyglycosides in an alternating fashion.3,4 Perhaps the best known member of the GAG-family is heparin, containing both D-glucuronic acid and L-iduronic acid moieties, that are interspaced with Nacetyl-D-glucosamine residues.5 Other GAGs are the hyaluronan6 (assembled of D-glucuronic acid and N-acetyl-D-glucosamine) and the chondroitin7 (D-glucuronic acid and N-acetyl-Dgalactosamine) polysaccharides. A structurally and functionally distinct class of polysaccharides are the so-called homoglycuronans, which contain only uronic acid residues. Examples are alginate8 (composed of D-mannuronic acid and L-guluronic acid) and pectin9,10 (D-galacturonic acid), both of which are often used in food industry. 9   

9

Chapter 1   

The structural complexity of uronic acid containing polysaccharides, combined with their diverse biological properties, has inspired many research groups to study their chemical synthesis. In general, the aim of these studies is the development of methodologies to introduce the required interglycosidic linkages and to apply these to construct oligosaccharides of a defined length and substitution pattern. These in turn are used in structure-function studies to determine the structural features that are at the basis of their biological properties. The potential of this general strategy is best illustrated by the extensive work on heparan sulfate, which led to the identification of a unique pentasaccharide sequence, which is at the basis of the anti-blood coagulation properties that characterize the natural polysaccharide.3b,11 Ensuing combined efforts at the Organon (Van Boeckel and coworkers) and Sanofi laboratories (Petitou and coworkers) led to the development of a closely related pentasaccharide as a drug (Arixtra®) for the treatment of thrombotic disease. In this Chapter some general strategies for the synthesis of uronic acid containing oligosaccharides are discussed. The focus is on the preparation and incorporation of the uronic acid building blocks.12 Two distinct strategies can be recognized. In most literature examples a target oligosaccharide is assembled from aldose building blocks, after which the appropriate primary hydroxyls are oxidized to carboxylates prior to or after global deprotection (post-glycosidation oxidation). The alternative general strategy entails the use of uronic acid building blocks in the glycosylation scheme (pre-glycosidation oxidation). Examples of both strategies will be presented, with a focus on the advantages and disadvantages of the respective strategies.

Post-glycosidation Oxidation The most frequently used method for the construction of acidic oligosaccharides is the initial construction of the oligosaccharide followed by oxidation of (specific) primary hydroxyl groups to the desired carboxylic acid functionality. Following this postglycosidation approach, Ogawa and coworkers investigated the synthesis of α-(1→4)dodecagalactosiduronic acid extracted from plant cell wall isolates (Scheme 1).13,14,15 Starting from the fluoride donors 1 and 3 and acceptor 2, dodecasaccharide 4 was assembled in a straightforward manner using the Mukaiyama coupling conditions (SnCl2, AgClO4).16 All glycosylation reactions proceeded predominantly α-stereoselective, which was attributed to attack of the acceptor nucleophile from the α-side and coordination of the diethyl ether solvent shielding the β-face. 17,18 The secondary alcohol groups were masked as benzyl ethers whereas the primary positions destined for oxidation were capped with acetyl groups. After construction of dodecasaccharide 4, selective liberation of the primary hydroxyl groups by treatment with base gave dodeca-ol 5 in good yield. Swern oxidation19,20 of dodecasaccharide 5 to the intermediate aldehyde and further oxidation with a freshly prepared solution of 10   

10

General Introduction   

NaClO2 in water afforded expected dodecacarboxylic acid 6 in 50% overall yield. Deprotection and purification afforded dodecasaccharide 7.

Scheme 1.

BnO

O

B nO

A cO O BnO 1

BnO Bn O

HO

O Ac

O

F

BnO

OR O BnO BnO

AcO O B nO O 2

BnO

B nO O

OR

10

1) S wer n o x. 2) NaClO2, 50%

O Bn O O Bn O

HO O COO H O B nO O

OTBDPS

B nO

F OBn

BnO HO

OR

4: R=A c 5: R=H

O

3

BnO NaOMe MeOH, 81%

AcO O BnO

COOH O BnO

O

Bn O OBn

Bn O

BnO

O

O

OTBDP S

O

BnO

OBn

O

OAc

COOH O HO

10

deprotection HO

O

HO COOH O

O TBDPS

HO

BnO 6

10

COO H O O

COOH O HO

OH

7

More recently, the group of Madsen published a procedure towards defined tri- and hexasaccharide fragments of the homogalacturonans.21 Here, orthogonality between the primary and secondary alcohol positions was conferred by application of paramethoxyphenyl (pMP) and benzyl protective groups. For the oxidation of all primary positions the two-step Dess-Martin periodinane22/NaClO2 protocol was employed. Compared to the Swern/NaClO2 protocol used by the group of Ogawa, lower oxidation efficiencies were found especially in the case of larger oligosaccharides. In a related study to elucidate the cleavage pattern of pectic enzymes, the group of Madsen embarked on the synthesis of defined, partly methyl-esterified fragments of the homogalacturonan polysaccharide.23 This pectic polysaccharide forms the primary cell wall matrix of all land plants and contributes to both the physical integrity and physiological status of the cell walls.24 Homogalacturonan is thought to be deposited in cell walls in a highly methyl-esterified form but can subsequently be de-esterified by pectin methyl esterases. Hence, the functionality of the pectin polysaccharide is largely determined by the pattern and degree of methyl esterification of the galacturonic acid backbone. In order to be able to introduce a partial methyl-esterification pattern in synthetic pectin oligosaccharides, the protective group strategy was slightly expanded. Acetyl protective groups were installed on the hydroxyl groups to be converted to methyl esters and para-methoxyphenyl protective groups were used for the hydroxyl groups to be converted to free carboxylic acids. By varying 11   

11

Chapter 1   

the acetyl and para-methoxyphenyl protection along the oligosaccharide backbone all the different partly methyl-esterified oligosaccharides can in theory be obtained. A typical example is depicted in Scheme 2 and commences with trisaccharide 8, which was synthesized in a straightforward manner using the n-pentenyl glycosylation technique. After deacetylation under standard conditions (8 → 9) the two-step Dess-Martin periodinane/NaClO2 oxidation protocol was applied. Reaction of the intermediate carboxylic acid with trimethylsilyldiazomethane afforded methyl ester 10. Subsequent cleavage of both paramethoxyphenyl ethers (11) and oxidation of the residual primary alcohol positions yielded trisaccharide 12 bearing orthogonally functionalized carboxylate groups. Final de-benzylation under standard conditions provided target trisaccharide 13. By applying the same orthogonal approach, hexasaccharide structures with varying patterns of methyl esters were synthesized.25

Scheme 2. BnO BnO

BnO

OpMP O BnO

O

OpMP O

BnO

BnO BnO

O

O

BnO 1) Dess-Martin 2) NaClO2 3) TMSCHN2

BnO

54%

BnO

RO O

OR

OBn

57%

BnO

8: R=Ac 9: R=H

O

COOH O RO RO

O

COOH O RO

COOMe O

OBn

RO

BnO

BnO K2CO3 MeOH, 70%

Dess-Martin then NaClO2

O BnO

OR O

RO

OR

CAN aq. MeCN, 70%

10: R=pMP 11: R=H

O

COOMe O RO

Pd/C, H2 MeOH/H2O

OR

12: R=Bn 13: R=H

Chromium based oxidation methods such as the strongly acidic Jones reagent26 and the milder pyridinium dichromate27 (Collins reagent, PDC) and pyridinium chlorochromate28 (PCC) reagents have also found application in carbohydrate chemistry. Depending on the reaction conditions, either the carboxylic acids or the corresponding carboxylic esters are isolated. Disadvantages of these chromium based oxidations are purification problems and restricted choice of protective groups.29 A typical example on the use of PDC comes from the group of Vliegenthart and Kamerling in their synthesis of a pentasaccharide fragment (17) of the gut-associated circulating anodic antigen (CAA, Scheme 3).30 After complete assembly of pentasaccharide fragment 14 using trichloroacetimidate chemistry,31 the C6-OH levulinoyl protective groups on both glucose residues were selectively cleaved giving compound 15. Subsequent oxidation using the chromium(VI) oxidant system (PDC, Ac2O, cat. pyridine) gave uronic acid 16. Acetic anhydride is added to facilitate cleavage of chromium(VI) from the intermediate ester, thereby accelerating the reaction.32 Global deprotection yielded pentasaccharide 17 in an overall yield of 65%. In a related study on the synthesis of smaller

12   

12

General Introduction   

fragments of pentasaccharide 17 several other oxidation protocols (including PCC, Jones and Swern protocols) proved to be less effective.33

Scheme 3. AcO

AcO

OAc O

AcO

O

AcO

HO

OAc O

O

OH O

HO

O

PhthN AcO OR TolO TolO

O TolO TolO TolO

PDC, Ac2O, PhthN AcHN cat. pyridine, AcO HO deprotection COOH COOH DCM O O O O O TolO HO O O 73% TolO O O 65% HO O O PhthN TolO PhthN HO AcHN AcO AcO HO OR COOH COOH O O O O O O TolO HO O OAll O OAll O TolO HO TolO PhthN TolO PhthN HO AcHN

NH2NH2·AcOH toluene/ethanol 92%

14: R=Lev 15: R=H

16

OAll

17

En route to the preparation of a pentasaccharide hapten of the Mycobacterium avium serovar 19, the group of Lipták explored the synthesis of uronates 20 and 23 (Scheme 4.).34 Regioselective de-acetylation of the L-manno containing disaccharide 18 proceeded in good yield giving compound 19, whereas subjection of L-talo equipped disaccharide 21 to the same conditions gave diol 22.35 Jones oxidation of the primary alcohol in disaccharide 19 afforded the intermediate glucuronide, which was transformed into the the methyl ester (20) by treatment with an ethereal diazomethane solution. In order to achieve oxidation of diol 22, the authors returned to a TEMPO-based oxidation method.36 Sodium hypochlorite was used as Scheme 4. OMe RO MeO MeO

MeO O

O O

OMe

1) CrO3, 3.5M H2SO4 acetone 2) CH2N2, Et2O, DCM 64%

OAc NaOMe 18: R=Ac MeOH, 86% 19: R=H

RO MeO MeO

O

MeO O

O

OMe

OR NaOMe 21: R=Ac MeOH, 95% 22: R=H

OMe MeOOC MeO O MeO O MeO OAc

O OMe

20 1) TEMPO, NaOCl, KBr NaHCO3, H2O 2) CH3I, DMF OMe 71%

MeOOC MeO MeO

O

MeO O

O

OMe

OMe

OH 23

co-oxidant, which reacts in situ with potassium bromide to generate the more reactive sodium hypobromite.37 Ensuing addition of methyl iodide and N,N-dimethylformamide (DMF) to the concentrated oxidation mixture afforded methyl ester disaccharide 23 in good yield. The 13   

13

Chapter 1   

choice for a different oxidation method was guided by the presence of the unprotected secondary C2’ hydroxyl group, which could also be oxidized using chromium(VI)-based oxidants.38 The finding that TEMPO is able to selectively oxidize primary hydroxyl functions in the presence of secondary hydroxyl groups has led to numerous applications of this reagent in oligosaccharide synthesis.36,39,40-48 Protective group manipulations to discriminate between primary and secondary hydroxyl groups have therefore become obsolete and orthogonality between the oxidized and the non-oxidized C6 positions is more easily attained. Depending on the amount of primary oxidant (co-oxidant) added and the reaction medium (anhydrous or aqueous) the oxidation reaction can be stopped at the aldehyde or carboxylic acid stage (Figure 1). Many primary oxidant systems have been reported including electro-oxidation,41 m-chloroperbenzoic acid,42 high-valent metal salts,43 sodium bromite,44 sodium or calcium hypochlorite,37,40a,45 hypervalent iodine(III) salts,46,47 and trichloroisocyanuric acid.48 The actual oxidizing species in all these reagent combinations is the N-oxoammonium intermediate 24 generated in situ from reaction between TEMPO and the primary co-oxidant (Figure 1).36,49 Anhydrous conditions give rise to the aldehyde whereas in the presence of water, the aldehyde is hydrated, allowing further oxidation to the carboxylic acid. Van Bekkum and coworkers postulated the formation of reaction intermediates 26 and/or 27 depending on the conditions used.40b,50

Figure 1. R-CH 2-OH

24

R-CHO

p roposed inte rmediates

H 2O R-CH(OH)2

a lkal ine me dium

RCOOH

acidic mediu m

co-oxidant -H

N

N

O

O

OH

TEMPO

24

25

N

r egener atio n (de pending on pH, see re f 36)

N

N O O

R

H R

HO O

1

R 26

2

1

R

2

H 27

B

En route towards synthetic fragments of the heparin polysaccharide, the group of Boons used TEMPO/NaOCl as a regioselective oxidation procedure (Scheme 5).51 Using trichloroacetimidate based glycosylation strategies trisaccharide 28 was obtained in good yields. In compound 28 the primary hydroxyl group in the glucosamine residues is protected as the tert-butyldiphenylsilyl (TBDPS)-ether. Oxidation of the two C6-OH functions (29) followed by desilylation gave target trisaccharide 30. It was reported that best selectivities were achieved when the reaction was performed under basic conditions at pH = 10.

14   

14

General Introduction    Scheme 5. OH HO HO

O OH

OTBDPS

TEMPO, NaOCl HO NaBr, NaOH, H2O HO

O

O HO

AcHN 28

OH O HO

O

O

COOH O OH

pH = 10 89% HF·pyridine aq. CH3CN 74%

OH

OR O

O HO

AcHN 29: R=TBDPS 30: R=H

O HO

COOH O

O

OH

As part of the construction of oligomeric structures corresponding to the capsular polysaccharide Streptococcus pneumoniae type 3, the group of Oscarson explored the synthesis of dimer 32 (Scheme 6).52 TEMPO-oxidation of the C6’ hydroxyl function of minimally protected disaccharide 31 gave target uronate 32 in only a moderate yield of 33%. As a side-product, substantial amounts of tri-uronate species 33 resulting from oxidative cleavage of the trans-diaxial diol (C2-OH C3-OH) system in compound 31 was isolated. Variation of the reaction conditions, such as other solvents and different pH-values met with similar failure. The unwanted side reaction was prevented by oxidizing partially protected disaccharide residues under biphasic dichloromethane (DCM)/H2O-conditions45a (see below) and subsequent deprotection.

Scheme 6. OH O

OH HO BnO

O OH 31

O

O

OH

1) TEMPO, NaOCl NaBr, NaOH, 4M HCl H2O, pH = 10 2) H+, MeOH 3) BzCl, pyridine

BzO BnO

COOMe O

OBz O O

OBz 32 (33%)

O

OBz

O + BzO BnO

O

COOMe O

O

COOMe COOMe

OBz 33

The group of Field studied the oxidation of di- (34), tri- (36) and tetrasaccharide (38) fragments of a rhamnogalacturonan-II polysaccharide using the TEMPO/NaOCl/KBr-reagent combination (Scheme 7).53 The presence of solely oxidized and/or deoxygenated C6 functionalities allows the oxidation of completely unprotected precursors 34, 36 and 38. The yield of the reactions decreased with the increasing complexity of the oxidation targets. This trend can be explained by the increased steric bulk, which is found to be an important factor in oxidation reactions under alkaline conditions (see Figure 1).36,40 Anelli and coworkers reported biphasic DCM/H2O as a highly suitable medium for TEMPO/NaOCl-mediated oxidations of partially protected oligosaccharides.45a It was revealed that under standard conditions, initial oxidation to the aldehyde is rather slow. Addition of the quaternary ammonium salt tetra-butylammonium bromide (TBABr) as a phase transfer catalyst considerably accelerates the oxidation rate. Furthermore, alkaline

15   

15

Chapter 1    Scheme 7. OH HO OMe HO

R

HO

HO O

O

OMe HO

O

HO

OH

R

HO O

OH

O

OH HO

34: R=CH2OH 35: R=COOH

TEMPO 73%

TEMPO 64%

HO

O O

R

OMe

O

HO HO

O

O O

O O

O

OH

R

HO

HO OH 36: R=CH2OH 37: R=COOH

O

R

HO OH 38: R=CH2OH 39: R=COOH

TEMPO 47%

conditions, such as aqueous NaHCO3, increases both reactivity and selectivity for primary alcohols (see Figure 1.).50a Flitsch and coworkers applied this method for the first time on protected monosaccharide residues.45b Petillo and coworkers reported on the NaOBr-mediated oxidation of the partly protected trisaccharide 40 en route to hyaluronan oligosaccharide 41 using the biphasic DCM/H2O-system (Scheme 8).54 Another example of this biphasic TEMPO-oxidation protocol is given by Litjens et al. in the synthesis of the repeating unit trisaccharide of the lysoamidase bacteriolytic complex.55

Scheme 8. HO HO HO

Ph

O O OH

O

O

O AcHN 40

BnO O HO

1) TEMPO, NaOCl KBr, TBABr, NaHCO3 OBn HOOC DCM/H2O (6/1), 74% OMe HO HO O 2) H2, Pd(OH)2 MeOH/H2O (10:1) 57%

OH O HO OH

O O AcHN

OH HO O HOOC

O

OMe

41

Recently, Huang and coworkers reported a two-step oxidation procedure for a partially protected hexasaccharadic hyaluronan fragment.56 Initial conversion of the substrate to the aldehyde using the TEMPO/NaOCl reagent combination is followed by further oxidation with sodium chlorite (NaClO2) to the corresponding uronic acid derivative. Increased lipophilicity of the substrate reduces the hydration rate of the intermediate aldehyde and thereby decreases the efficiency of the overall oxidation. This procedure, and in particular the use of NaClO2 in combination with tBuOH, is claimed to be less sensitive to changes in the hydrophobicity of the substrate molecule.

16   

16

General Introduction   

Pre-glycosidation Oxidation In pre-glycosidation oxidation strategies, suitably protected donor and/or acceptor glycuronates are employed in the construction of acidic oligosaccharides. The presence of the electron withdrawing ester function make these uronic acids less reactive compared to their non-oxidized counterparts. Donor uronic acid derivatives that have found application in oligosaccharide synthesis over the years include anomeric bromides,57 fluorides,58 orthoesters,59 trichloroacetimidates,60 n-pentenyl glycosides,61 and 1-thioglycosides.56 The group of Ogawa investigated the glycosylation properties of protected galacturonic acid fluorides in the synthesis of truncated pectic polysaccharides (Scheme 10).58 Fluoride donor 45 and acceptor allyl uronate 44 were synthesized from substrate 42 (Scheme 9). Jones oxidation not only led to a sluggish and incomplete reaction but also to migration of the chloroacetyl group to the thermodynamically favored C6 position. Catalytic oxidation using Pt/NaHCO3/H2O resulted in incomplete reactions. Swern oxidation of compound 42 followed by Jones oxidation of the intermediate aldehyde and subsequent treatment with ethereal diazomethane afforded uronic acid derivative 43 in a yield of 72%. Exchange of the anomeric allyl group (43) for a fluoride afforded uronate donor 45. Galacturonate acceptor 44 was obtained by treatment of fully protected compound 43 with thiourea. Takeda and coworkers also encountered problems during the synthesis of OH4-unprotected galacturonates and therefore opted for the post-glycosidation oxidation strategy in their synthesis of a pectic polysaccharide repeating unit.62

Scheme 9. ClAcO BnO

1) Swern oxidation 2) 8N Jones reagent 3) CH2N2, Et2O

OH O OBn 42

OAll

72%

RO BnO

thiourea EtOH, 70%

COOMe O

ClAcO from 43 OAll

OBn 43: R=ClAc 44: R=H

BnO

COOMe O BnO

F

45

Subjection of uronic acids 45 and 44 to Mukaiyama conditions (SnCl2, AgClO4)16 did not result in a productive glycosylation reaction, most probably due to the deactivating influence of the remotely attached uronic acid esters (Scheme 10). Indeed, application of galactosyl fluoride 46 as a more reactive donor gave α-linked disaccharide 48 in a yield of 42%. The yield was further improved by using non-oxidized, acetyl protected acceptor 47 instead, giving α-linked disaccharide 49 in 83% yield. From these results it can be concluded that the use of anomeric fluorides in combination with the Mukaiyama activation conditions is less suitable in the pre-glycosidation oxidation approach.

17   

17

Chapter 1   

Vogel and coworkers investigated the trityl-cyanoethylidene glycosidation method63,64 for the construction of acidic oligosaccharides.65 This method proved to be successful in the synthesis of β-(1→2)- and β-(1→3)-linked digalacturonic acid residues. The construction however, of the demanding β-(1→4)-linked dimers resulted in complex reaction mixtures with minor product formation.66

Scheme 10. ClAcO BnO

HO

COOMe O BnO 45

ClAcO

+

46

+ F

SnCl2, AgClO4 OAll

OBn 44 HO

O BnO

COOMe O

F

OAc

BnO

BnO

BnO

R

no reaction

Et2O, -15°C

ClAcO O

SnCl2, AgClO4 OAll

OBn 44: R=COOMe 47: R=CH2OAc

Et2O, -15°C

BnO

OAc OAll

O O BnO

OR

OBn

BnO 48: R=COOMe (42%) 49: R=CH2OAc (83%)

Westman and coworkers reported on the use of anomeric bromides and 1-thioglycuronides in the synthesis of defined fragments of a known glycosaminoglycan tetrasaccharide.67 In order to minimize protective group manipulations a block type synthesis strategy was chosen in combination with an orthogonal12e glycosylation strategy. Starting from peracetylated methyl glucuronate 50 the corresponding iduronic acid68 donor 51 was obtained using a radical initiated epimerization around C5 (Scheme 11).57a,b Iduronic acid 51 was then converted into bromide donor 52 upon treatment with a hydrogen bromide (HBr)-solution in acetic acid. Thio donor 54 was obtained by oxidation of compound 53 with PDC and acetic anhydride in a mixture of tert-butanol and DCM.69 In this process, the intermediate aldehyde is trapped by tert-butanol to give the tert-butyl hemiacetal, which is further oxidized to give tert-butyl uronate 54. No oxidation of the thiophenyl function to the corresponding sulfoxide or sulfone is reported. In an orthogonal glycosylation strategy, iduronic acid bromide 52 was condensed with ethylthio glucosazide 55 giving 1-thiodisaccharide 57 in a yield of 60%. The 1thioglucuronide 54 was glycosylated with acceptor 56 under the agency of dimethyl(methylthio)sulfonium triflate (DMTST) giving compound 58.70 The synthesis of tetrasaccharide 60 was completed by oxidative cleavage of the para-methoxybenzyl (pMB) group (58 → 59) and DMTST-mediated coupling between thiodisaccharide 57 and glucuronide acceptor 59. This approach highlights the usefulness of the stable 1thioglucuronides as both donor and acceptor in acidic oligosaccharide synthesis.

18   

18

General Introduction    Scheme 11. 2

R

R3 R1 OAc O

OBn

ref. 57

MeOOC

OAc O

SEt

SEt

PDC, tBuOH DCM, 72%

55

AgOTf, DTBMP DCM, 60% N3 BnO SEt O O

RO BnO

COOtBu O

56

N3 O

AcO

COOtBu O

O BnO

OBn O

OMe

O OBn

58: R=pMB 59: R=H

OBn O

O

OMe

OBn

OBz

OBn

AcO

OBz CAN, ACN 80%

MeOOC

OBn

53: R=CH2OH 54: R=COOtBu

DMTST DTBMP DCM, 79%

OAc

OAc O

OMe

HO

DMTST, DCM 0°C, 80%

OBn

BnO O

OBn O

+

OBz

57 AcO

O

pMBO BnO

N3

AcO OAc 50: R1=COOMe, R2=H, R3=OAc 51: R1=H, R2=COOMe, R3=OAc 52: R1=H, R2=COOMe, R3=Br

HBr/AcOH

AcO

R

O

HO BnO

+

60 AcO

OAc

Another successful application of 1-thioglycuronic acid esters in acidic oligosaccharide synthesis was published by the group of Robert-Baudouy in their synthesis of defined fragments of the pectic polysaccharide.71,72 Chemoselective oxidation of the partially protected 1-thioglycoside 61 was accomplished using the PDC/Ac2O/tert-BuOH oxidation system (Scheme 12.). Exchange of the acid labile tert-butyl ester in compound 62 for either a benzyl or a methyl ester yielded galacturonates 63 and 64. The benzyl and methyl esters were introduced under acidic conditions to prevent putative β-elimination or epimerization reactions, which were earlier observed by the groups of Sinaÿ73 and Vogel.73,74,75 Direct application of the tert-butyl ester functionalized glycosides sometimes resulted in compromized coupling efficiencies due to the increased steric bulk.76 It was found that application of the N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH) activator system77 is successful in the α-stereoselective glycosidation of these deactivated galacturonate building blocks with acceptor building blocks 65 and 66 (Scheme 12.).

Scheme 12. BnO

PDC, Ac 2O ter t-B uOH

OH O SPh

Bn O OBn 61

70%

BnO

COOtBu O

20% TFA in DCM SPh

Bn O OBn 62

BnB r, NaHCO 3 TBA I, DCM/H 2O or A cCl, MeOH

BnO

HO

CO OR O SPh

BnO OBn

63: R=Bn (6 8%) 64: R=Me (96% )

COO R O OBn

BnO OBn 65: R=Bn 66: R=Me

19   

19

Chapter 1   

Sinaÿ and coworkers published a study in which the glycosylation properties of n-pentenyl 70, trichloroacetimidate 71, and 1-thio (75 + 76) and functionalized iduronic acid donors were compared (Scheme 13).61 Starting from 6-O-tert-butyldimethylsilyl protected 67 and 68, uronic acid esters 69 and 70 were obtained using Jones reagent (CrO3, 3.5M H2SO4) and subsequent methylation. Disaccharides could also be oxidized by this one-pot silyl cleavage/oxidation protocol although the yields dropped slightly.71 After oxidation, 1-Oacetyl derivative 69 was transformed into the corresponding trichloroacetimidate donor 71 via a known sequence of reactions (α-Ac → α-Br → α/β-OH → α/β-OC(NH)CCl3). Jones oxidation of the corresponding 6-O-TBDMS functionalized phenylthio residue 72 proceeded less straightforward and, along with the desired 1-thio-α-L-iduronic acid ester 75 (26%), considerable amounts of sulfoxide and sulfone were isolated (together ~50%).78 Acidic cleavage of the silyl group (72 → 73) and oxidation using the milder pyridinium dichromate (PDC) gave a substantially improved yield of 1-thio iduronic acids 75 and 76 (both 52%). Table 1 summarizes the glycosidations performed with the donors 70 and 71. It can be concluded that trichloroacetimidate 71 and n-pentenyl glycoside 70 are equally efficient in glycosylating acceptors 78 and 79. On the other hand, the corresponding 1-thioiduronates 75 and 76 did not yield the expected disaccharides using DMTST70 as activator.79 Closer inspection revealed that thiophenyl donor 75 was completely inert for DMTST activation whereas thioethyl donor 76 showed minor formation of lactone 77.

Scheme 13. NH TBDPSO

LevO

OBn O

OR1

CrO3, 3.5M H2SO4 acetone then MeI, KHCO3, DMF

OR2

LevO

67: R1=R2=Ac 68: R1=n-pentenyl, R2=Bz

R2O

LevO Dowex H+ MeOH

OBn O

SR1

MeOOC

PDC, DMF then MeI, KHCO3, DMF

OBz

72: R1=Ph, R2=TBDMS 73: R1=Ph, R2=H 74: R1=Et, R2=H

OBn O

OR1 from 69

OR2

MeOOC

OBn O

LevO

LevO

OBn O

CCl3

OAc

69: R1=R2=Ac (54%) 70: R1=n-pentenyl, R2=Bz (55%)

MeOOC

O

71

SR

OBz

75: R=Ph (52% from 73) 76: R=Et (52%)

O

LevO BnO

AcO O

O 77

The groups of Garegg and Oscarson investigated the use of 1-thioglycuronides in their study towards the synthesis of naturally occurring acidic polysaccharides isolated from Streptococcus pneumoniae and Cryptococcus neoformans.80 In agreement with the observations of Ogawa and coworkers,81 Lewis acid-mediated reaction between ethanethiol

20   

20

General Introduction    Table 1.

entry

donor

activator yield (α/β)

acceptor

disaccharide

NH

1

OBn O

Me OOC L evO

N3 CCl3

O

OAc

TMSOTf 91% (1/0)

O

HO BnO

N3

OAc

MeO OC

OBn O

LevO

OMe

O O Ac

N3 O

2

OBn O

MeOO C LevO

NIS/TfOH 80% (1/0)

78

MeO OC

OBn O

LevO

O Bz

B nO O

O O Ac

81 A cO AcO

71

O HO

OBn O

OBn

N3

OMe

TMSOTf 86% (1/0)

Me OOC L evO

79

OBn O

O

OAc 82 OBn O

70

79

NIS/TfOH 85% (1/0)

MeOOC LevO

O Me

N3

AcO

4

OMe

O Bz

70

3

OMe

O Ac 80

78

71

B nO O

OBn O

O

OMe

N3

OBz 83

and peracetyl methyl (β-D-glucopyranoside) uronate resulted in a moderate yield and low stereoselectivity. It was therefore decided to examine direct oxidation of suitably protected ethyl 1-thioglycoside 84 (Scheme 14). Oxidation was accomplished using a two-step oxidation protocol comprising first Pfitzner-Moffat oxidation82 of alcohol 84 to the intermediate aldehyde and ensuing treatment with excess PDC giving donor 86 in a yield of 71%. In the same way, 2-O-acyl donor 87 was prepared from compound 85. Furthermore, basic hydrolysis of the 2-O-acetate (87 → 88) and reprotection yielded donor species 89, 90 and 91. The glycosylation properties of donor 86, having a C2 benzyl group and donors 87, 89, 90 and 91, having a C2 acyl group were investigated using acceptors 92 and 93 and DMTST as system. DMTST-mediated coupling of tri-O-benzyl 1-thioglucuronide 86 with acceptors 92 and 93 afforded the corresponding disaccharides 94 and 99 as anomeric mixtures. In a later study by the same group it was found that 2-O-benzylated 1thioglucuronide donors are sensitive to changes in reaction conditions, such as different promoter systems, different protective groups, and application of a participating solvent.83 This was independently proven by a study of Misra and Roy, who reported complete α-

21   

21

Chapter 1   

selectivity using tribenzylated donor 86 in methyl triflate-mediated glycosylations.84 On the other hand, glycosylations using 2-O-acylated donors 87 and 91 proved problematic whereas 2-O-benzoylated derivatives 89 and 90 showed good coupling efficiencies.

Scheme 14. OH

1) DMSO, DCC pyridine, TFA

O

BnO BnO

SEt

2) PDC, MeOH DMF

OR 84: R=Bn 85: R=Ac

OH O

O

O BnO 92

BnO BnO

O

O

O BnO

OMe

O

SEt

BnO BnO

COOMe O

OMe

86: R=Bn 87: R=Ac 89: R=Bz 90: R=MBz 91: R=Piv

94: R=Bn (76%, α/β=2/1) 95: R=Ac (25%, β) 96: R=Bz (68%, β) 97: R=MBz (65%, β) 98: R=Piv (31%, β)

or PivCl, pyr.

BnO BnO

COOMe O

SEt

OR 89: R=Bz (90%) 90: R=MBz (86%) 91: R=Piv (85%)

DMTST, DCM, MS4A O OBn O

SEt

OR

COOMe O OR O

Ph

BzCl, pyr. or MBzCl, pyr.

OR 86: R=Bn (71%) 87: R=Ac (68%) 88: R=H

NaOMe MeOH, 70%

DMTST, DCM, MS4A Ph

MeOOC BnO BnO

HO 93 OBn COOMe O BnO BnO 99: R=Bn (69%, α/β=2/1) OR O 100: R=Ac (33%, β) 101: R=Bz (69%, β) BnO 102: R=MBz (60%, β) 103: R=Piv (53%, β)

OBn O O

Van der Marel and coworkers recently published on the use of the TEMPO/ [bis(acetoxy)iodo]benzene (BAIB) reagent combination for the efficient oxidation of variously functionalized 1-thioglycosides (Figure 2).85,86 Both the starting glycoside (glucose, glucosamine, galactose, and idose) and the nature of the protective groups (benzyl, benzoyl, isopropylidene, tert-butyldimethylsilyl, azide, and phthalimide) can be varied without major implications on the outcome of the oxidation step. Furthermore, the mild oxidation conditions allow the presence of unprotected secondary hydroxyl functions and various substituted 1-thio functions. Subjection of 4,6-unprotected thioglycosides 104 to the TEMPO/BAIB oxidation Figure 2. OH HO

O

cat. TEMPO BAIB SR

(OP)n

DCM/H2O (2/1)

HO

COOH O

104

CH2N2 SR

HO

COOMe O

(OP)n

(OP)n

105

106

SR

O OH HO

O (OP)n 107

22   

22

cat. TEMPO BAIB SPh

DCM/H2O (2/1)

SPh

O (PO)n

O

H+, MeOH HO

COOMe O (OP)n

108

109

SPh

General Introduction    85

conditions afforded the corresponding uronic acids 105 whereas 3,6-unprotected thioglycosides 107 resulted in a tandem oxidation/lactonization process giving the corresponding 6,3-lactones 108.86 Methyl ester 106 was obtained upon treatment of uronic acid 105 with ethereal diazomethane, and acidic cleavage of lactone 108 in MeOH gave methyl ester 109. In a related study using the TEMPO/NaClO2 oxidation system, Huang and coworkers also reported on the chemo- and regioselective oxidation of variously protected 1thiotolylglycosides.56

Figure 3. O S

OTf R

Tf2O

S

110

a: R = piperidino (BSP) b: R = phenyl (Ph2SO)

R OTf

-60°C 111

The donor and acceptor properties of uronates 106 and 108 were investigated using the BSP (110a)/Tf2O87 or Ph2SO (110b)/Tf2O88 activator system (Figure 3). It was assumed that the electrophilic nature of these activator systems was sufficient to overcome the reduced nucleophilicity at the anomeric centre due to the remotely attached carboxyl function. Indeed, both activator systems proved successful in the glycosidation of oxidized gluco- and galactopyranosides, although activation had to be performed at slightly higher temperature (-40°C – -50°C) compared to the standard procedure. Comparison of the methyl uronate 112 and lactone 115 shows the latter to be more αselective in glycosidation reactions with the same acceptor molecule 113 (Table 2, entries 1 and 2). Even fully acylated donor 117 was efficiently glycosidated with acceptor 113 under the agency of the BSP/Tf2O reagent combination (entry 3). Illustrative in this respect are the examples published by the groups of Garegg and Oscarson who had to install at least two activating benzyl functions or a 3,4-tetraisopropyldisiloxyl group at the thioglucuronate donors (for instance 89, Scheme 14.) to obtain sufficient activation with DMTST (see Scheme 14).80 N-(benzyloxycarbonyl)-protected glucosamine 119 was efficiently employed as acceptor nucleophile giving, with full α-selectivity, disaccharide 120 (entry 4). This reaction proceeded slower than the other glycosidations, possibly due to reduced reactivity of the acceptor alcohol via intramolecular H-bonding.89 Glycosidations using the 1-thiomannuronic acid ester donors (e.g. 121) have different outcomes compared to their 1-thioglucuronic and galacturonic acid ester congeners (entry 5). The gluco- and galacto-configured uronate donors show preference for α-product formation, almost all the mannuronic acid derivatives exclusively afford the β-oriented product (see Chapter 4).90 In analogy with the findings of

23   

23

Chapter 1    Table 2.

entry

donor

acceptor

activator yield (α/β)a,d

disaccharide AcO

AcO

1

OH

COOMe O

SPh

BnO

O

BnO BnO

BnO OMe 113

OBn 112

b

Ph2SO/Tf2O 80% (1/2)

BnO

SPh

O BnO

AcO BzO

O O

BnO

AcO BzO

b

SPh

BSP/Tf2O 68% (0/1)

113

OBz

BnO OBn OBn 116

COOMe O

HO BnO

115

CbzHN 119

5

MeOOC OBn O AcO BnO 121

a

SPh

Ph2SO/Tf2O 69% (1/0)

OBn

OBn

O BnO

Ph2SO/Tf2O 81% (0/1)

O

O BnO

O

OBn 120

b

113

BnO

O c

OMe

OMe OBn

O

O

OBz

OBn O

OMe OBn

O

118

4

BnO OMe

O

OBn

COOMe O 117

Ph2SO/Tf2Ob 69% (1/0)

113

O 115

3

O

O BnO BnO 114

O

O

2

COOMe O

BnO

MeOOC OBn O AcO BnO

O

BnO 122

CbzHN

O

OMe

OMe OBn

OBn

isolated yields b 2.5 equiv. TTBP c 1 equiv. TTBP d anomeric ratios were determined by 1H NMR spectroscopy.

Crich91 and Bols,92 it is assumed that the strong electron-withdrawing nature of the remotely attached carboxyl group combined with the enlarged anomeric effect in the manno-series dictates the reaction towards the β-product. Scheme 15 displays the synthetic strategy for the assembly of the protected carbohydrate motif GlcpNAcα(1→4)-GlcpAα(1→2)Galf (127) present in the capsular polysaccharide of the Fungus Fusarium sp. M7-1.93 Activation of hemiacetal donor 123 with diphenylsulfonium bistriflate 111b in the presence of tri-tert-butylpyrimidine (TTBP)94 as a base and subsequent treatment with uronic acid acceptor 124 gave the α-linked thiodisaccharide 125 as the sole product in 67% yield (Scheme 3). In the second glycosylation event, disaccharide 125 was activated under the agency of the BSP/Tf2O activator and condensed with partially protected 24   

24

General Introduction   

galactono-1,4-lactone 126 to give trisaccharide 127 as an anomeric mixture (α/β = 4/1) in a yield of 35%. Based on a related iterative, chemoselective glycosylation approach, Codée et al. described a modular synthesis approach towards a defined heparin pentasaccharide starting from pre-oxidized glucuronic and iduronic acid building blocks.95

Scheme 15. OAc O

BnO BnO

HO BnO

+

Ph2SO, Tf2O TTBP, DCM

COOMe O

N3 OH

SEt

OBn

123

-40°C, 67%

OAc

124

125

OAc

N3 O BnO

COOMe O

SEt

OBn

O

O

BnO BnO

O

BnO BnO

O

COOMe BzO O

N3 O BnO

-60°C, 35% α/β=4/1

O

O

BnO

127

BSP, Tf2O TTBP, DCM

H

O

H

O

O

O

BzO 126 OH

O

Scheme 16 depicts the strategy for the first synthesis of alginate trisaccharide 134.90 The synthesis starts from the non-reducing end monosaccharide 128 and makes use of levulinoyl groups as temporary protection. Unmasking of the Lev-group in disaccharide 130 afforded the new acceptor 131 which was condensed with donor 128 to give trisaccharide 132 in 50% yield. Global deprotection afforded alginate trisaccharide 133, which was recently reported as a potential ligand for Toll-like receptors 2 and 4. In the same way, 1-thio functionalized mannosaziduronic acid showed promise as donor and acceptor glycosides in carbohydrate chemistry (see Chapter 5).96 Scheme 16. MeOOC OBn O LevO BnO 128 SPh

3

NIS/TMS OTf DCM, -40°C (α /β = 1/>10)

Me OOC OBn O HO B nO

O N3

129

1

R O OC O R O RO 1 R O 2

de protection 35%

3

NH 2NH2, A cOH pyridine , 95%

1

R OO C OR O O1 RO 1

MeOOC OBn O RO BnO

78 %

NIS/TMSOTf DCM, -40°C, (α / β = 1/>1 0)

1

3

R OO C OR O O R1O 2

3

O 4

R

4

Me OOC OBn O O B nO

O N3

130: R=Lev 131: R=H MeOOC OBn O LevO BnO 128 SPh

50 %

132 : R =Bn, R =Lev, R =Me, R =N3 1 2 3 4 133 : R =R =R =H, R =NH 3O Ac

25   

25

Chapter 1   

Conclusion This Chapter reviews the developments in the synthesis of acidic oligosaccharides. Both approaches for the introduction of uronic acid residues (that is, oxidation prior to or post glycosylation) in acidic oligosaccharides are discussed and typical examples are presented. It can be concluded that the post-glycosylation strategy requires an additional protective group manipulation and risks of losing valuable oligosaccharide compound during oxidation. The pre-glycosylation protocol avoids these difficulties although the reactivity at the anomeric centre is impaired compared to the non-oxidized counterparts. In general, the advent of TEMPO as oxidizing reagent largely stimulated the development of new and efficient ways for the synthesis of acidic oligosaccharides. In comparison to the other, more robust oxidation methods, the applied protective group pattern is only minimally restricted allowing the oxidation of partially or completely deprotected carbohydrate structures. Furthermore, it has inspired the development of techniques to employ oxidized monosaccharide residues as building blocks in acidic oligosaccharide synthesis. Especially, application of the versatile thiogluronic acid esters holds promise in the synthesis of complex acidic oligosaccharides.

References and Notes 1 2

Lindberg, B.; Kenne, L. The Polysaccharides, Aspinall G.O. ed., Academic Press, New York, 1985. Lindberg, B. Adv. Carbohydr. Chem. Biochem. 1990, 48, 279–318.

3

(a) Thunberg, L.; Bäckstrom, G.; Lindahl, U. Carbohydr. Res. 1982, 100, 393–410 (b) Petitou, M.; Van Boeckel, C.A.A. Angew. Chem. Int. Ed. 2004, 43, 3118–3133. (a) Lindahl, U.; Kusche-Gullberg, M.; Kjellén, L. J. Biol. Chem. 1998, 273, 24979–24982 (b) Esko, J.D.; Lindahl, U. J. Clin. Invest., 2001, 108, 169–173. (a) Capila, I.; Linhardt, R.J. Angew. Chem. Int. Ed. 2002, 41, 391–412 (b) Hacker, U.; Nybakken, K.;

4 5 6 7 8

Perimon, N. Nat. Rev. Mol. Cell Biol. 2005, 6, 530–541. Laurent, T.C.; Fraser, J.R.E. FASEB J. 1992, 6, 2397–2404. Gama, C.I.; Tully, S.E.; Sotogaku, N.; Clark, P.M.; Rawat, M.; Vaidehi, N.; Goddard, W.A.; Nishi, A.; Hsieh-Wilson, L.C. Nat. Chem. Biol. 2006, 2, 467–473. Moe, S.T.; Draget, K.I.; Skjak-Braek, G.; and Smidsrød, O. In Food Polysaccharides and Their

Applications, Stephen A.M. ed., Marcel Dekker, Inc., New York, 1995, 245–286. Mohnen, D. In Comprehensive Natural Products Chemistry, Barton, D. Nakanishi, K.; Meth-Noho, O. eds, Elsevier Science Publishers BV, Amsterdam, 1999, Vol 3, 497–527. 10 Modifications bearing rhamnose residues are known: Ridley, B.L.; O’Neill, M.A.; Mohnen, D.A. 9

Phytochemistry 2001, 57, 929–967. 11 Bauer, K.A.; Hawkins, D.W.; Peters, P.C.; Petitou, M.; Herbert, J.-M.; Van Boeckel, C.A.A. Cardiovasc. Drug Rev. 2002, 20, 37–52. 12 See for reviews on the introduction of glycosidic linkages: (a) Paulsen, H. Angew. Chem. Int. Ed. Engl. 1982, 21, 155–224 (b) Boons, G.-J. Tetrahedron 1996, 52, 1095–1121 (c) Garegg, P.J. Adv. Chem. Biochem. 1997, 52, 178–205 (d) Davis, B.G. J. Chem. Soc., Perkin Trans. 1 2000, 2137–2160 (e) Codée, J.D.C.; Litjens,

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26

General Introduction    R.E.J.N.; Van den Bos, L.J.; Overkleeft, H.S.; Van der Marel, G.A. Chem. Soc. Rev. 2005, 34, 769–782 (f) Demchenko, A.V. Lett. Org. Chem. 2005, 2, 580–589. 13 Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1989, 30, 87–90. 14 Other studies of the group of Ogawa on the synthesis of α-(1→4)-linked homogalacturonans: (a) Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1987, 167, C1–C7 (b) Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1989, 194, 95–114 (c) Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1990, 200, 363–375 (d) Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1990, 205, 147–159. 15 Hahn, M.G.; Darvill, A.G.; Albersheim, P. Plant Physiol. 1981, 68, 1161–1169. 16 Mukaiyama, T.; Murai, Y.; Shoda, S. Chem. Lett. 1981, 431–432. 17 Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1987, 28, 2731–2734. 18 Ethers are known to be participating solvents: (a) Demchenko, A.V.; Stauch, T.; Boons, G.-J. Synlett 1997, 818–820 (b) Demchenko, A.V.; Rousson, E.; Boons, G.-J. Tetrahedron Lett. 1999, 40, 6532–6526. 19 For a comprehensive review on activated dimethylsulfoxide see: Mancuso, A.J.; Swern, D. Synthesis 1981, 165–185. Selected examples: (a) Omura, K.; Swern, D. Tetrahedron 1978, 34, 1651–1660 (b) Slaghek, T.M.; Nakahara, Y.; Ogawa, T. Tetrahedron Lett. 1992, 33, 4971–4974 (c) Slaghek, T.M.; Nakahara, Y.; Ogawa, T.; Kamerling, J.P.; Vliegenthart, J.F.G. Carbohydr. Res., 1994, 255, 61–85 (d) Slaghek, T.M.; Hyppönen, T.K.; Ogawa, T.; Kamerling, J.P.; Vliegenthart, J.F.G. Tetrahedron Lett. 1993, 34, 7939–7942 (e) Slaghek, T.M.; Hyppönen, T.K.; Ogawa, T.; Kamerling, J.P.; Vliegenthart, J.F.G. Tetrahedron Asymm. 1994, 5, 2291–2301 (f) Lucas, R.; Hamza, D.; Lubineau, A.; Bonnaffé, D. Eur. J. Org. Chem. 2004, 2107–2117. 20 Failed oxidations using this protocol have also been published: Halkes, K.M.; Slaghek, T.M.; Hyppönen, T.K.; Kruiskamp, P.H.; Ogawa, T.; Kamerling, J.P.; Vliegenthart, J.F.G. Carbohydr. Res. 1998, 309, 161– 174. 21 Clausen, M.H.; Madsen, R. Carbohydr. Res. 2004, 339, 2159–2169. 22 (a) Dess, D.B.; Martin, J.C. J. Am. Chem. Soc. 1991, 113, 7277–7287 (b) Chambers, D.J.; Evans, G.R.; Fairbanks, A.J. Tetrahedron 2005, 61, 7184–7192. 23 Clausen, M.H.; Jørgensen, M.R.; Thorsen, J.; Madsen, R. J. Chem. Soc., Perkin Trans. 1 2001, 543–551. 24 Willats, W.G.T.; Orfila, C.; Limberg, G.; Buchholt, H.C.; Van Alebeek, G.-J.W.M.; Voragen, A.G.J.; Marcus, S.E.; Christensen, T.M.I.E.; Mikkelsen, J.D.; Murray, B.S.; Knox, J.P. J. Biol. Chem. 2001, 276, 19404–19413. 25 Clausen, M.H.; Madsen, R. Chem. Eur. J. 2003, 9, 3821–3832. 26 Bowden, K.; Heilbron, I. M.; Jones, E. R. H. J. Chem. Soc. 1946, 39–45. Selected examples of the use of the Jones reagent system in oligosaccharide systems: (a) Kováč, P. Carbohydr. Res. 1973, 31, 323–330 (b) Jacquinet, J.-C.; Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Torri, G.; Sinaÿ, P. Carbohydr. Res. 1984, 130, 221–241 (c) Betanelli, V.I.; Ott, A.Y.; Brukhova, O.V.; Kochetkov, N.K. Carbohydr. Res. 1988, 179, 37–50 (d) Carter, M.B.; Petillo, P.A.; Anderson, L.; Lerner, L.E. Carbohydr. Res. 1994, 258, 299–306 (e) La Ferla, B.; Lay, L.; Guerrini, M.; Poletti, L.; Panza, L.; Russo, G. Tetrahedron, 1999, 55, 9867–9880 (f) Mukhopadhay, B.; Roy, N. Carbohydr. Res. 2003, 338, 589–596. 27 (a) Coates, W.M.; Corrigan, J.R. Chem. Ind. 1969, 44, 1594–1595 (b) Corey, E.J.; Schmidt, G. Tetrahedron Lett. 1979, 20, 399–402. 28 Piancatelli, G.; Screttri, A.; D’Auria, M. Synthesis 1982, 245–258. 29 Palmacci, E.R.; Seeberger, P.H. Tetrahedron 2004, 60, 7755–7766. 30 Vermeer, H.J.; Halkes, K.M.; Van Kuik, J.A.; Kamerling, J.P.; Vliegenthart, J.F.G. J. Chem. Soc., Perkin Trans. 1 2000, 2249–2263. 31 Schmidt, R.R.; Castro-Palomino, J.C.; Retz, O. Pure Appl. Chem. 1999, 71, 729–744.

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Chapter 1    32 Garegg, P.J.; Samuelsson, B. Carbohydr. Res. 1978, 67, 267–270. 33 Halkes, K.M.; Vermeer, H.J.; Slaghek, T.M.; Van Hooft, P.A.V.; Loof, A.; Kamerling, J.P.; Vliegenthart, J.F.G. Carbohydr. Res. 1998, 309, 175–188. 34 Fekete, A.; Gyergyói, K.; Köver, K.E.; Bajza, I.; Lipták, A. Carbohydr. Res. 2006, 341, 1312–1321. 35 (a) Szurmai, Z.; Lipták, A.; Carbohydr. Res. 1982, 107, 33–41 (b) Szurmai, Z.; Lipták, A.; Snatzke, G. Carbohydr. Res. 1990, 200, 201–208. 36 De Nooy, A.E.J.; Besemer, A.C.; Van Bekkum, H. Synthesis 1996, 1153–1174. 37 Anelli, P.L.; Biffi, C.; Montanari, F.; Quici, S. J. Org. Chem. 1987, 52, 2559-2562. 38 Jones oxidiation protocol: (a) Assarsson; Theander, O. Acta Chem. Scand. 1958, 1507–1509. Methods based on PDC-mediated oxidation of C2 position: (b) Wood, W.W.; Rashid, A. Tetrahedron Lett. 1987, 28, 1933– 1936 (c) Rashid, A.; Taylor, G.M.; Wood, W.W.; Alker, D. J. Chem. Soc., Perkin Trans. 1 1990, 1289–1296 (d) Perez-Perez, M.-J.; Camarasa, M.-J.; Diaz-Ortiz, A.; Felix, A.S.; De las Heras, F.G. Carbohydr. Res. 1991, 216, 399–411 (e) Lichtenthaler, F.W.; Immel, S.; Pokinskyj, P. Liebigs Ann. Chem. 1995, 1939–1948 (f) Hiraoka, S.; Yamazaki, T.; Kitazume, T. Synlett 1997, 669–670. 39 Maruyama, M.; Takeda, T.; Shimizu, N.; Hada, N.; Yamada, H. Carbohydr. Res. 2003, 325, 83–92. 40 (a) De Nooy, A.E.J.; Besemer, A.C.; Van Bekkum, H. Carbohydr. Res. 1995, 269, 89–98 (b) Söderman, P.; Widmalm, G. Eur. J. Org. Chem. 2001, 3453–3456. 41 (a) Semmelhack, M.F.; Chou, C.S.; Cortes, D.A. J. Am. Chem. Soc. 1983, 105, 4492–4494 (b) Inokuchi, T.; Matsumoto, S.; Torii, S. J. Org. Chem. 1991, 56, 2416–2421 (c) For a review, see: Yamaguchi, M.; Miyazawa, T.; Takata, Y.; Endo, T. Pure Appl. Chem. 1990, 62, 217–222 (d) Kashiwagi, Y.; Yanagisawa, Y.; Kurashima, F.; Anzai, J.; Osa, T.; Bobbit, J. M. Chem. Commun. 1996, 2745–2746. 42 (a) Cella, J.A.; Kelley, J.A.; Kenhan, E.F. J. Org. Chem. 1975, 40, 1860–1862 (b) Ganem, B. J. Org. Chem. 1975, 40, 1998–2000 (c) Cella, J.A.; McGrath, J.P.; Kelley, J.A.; El Soukkary, O.; Hilpert, L. J. Org. Chem. 1977, 42, 2077–2080. 43 (a) Miyazawa, T.; Endo, T. J. Mol. Catal. 1985, 31, 217–220. (b) Miyazawa, T.; Endo, T. J. Mol. Catal. 1985, 32, 357–360. (c) Semmelhack, M.F.; Schmid, C.R.; Cortes, D.A.; Chou, C.S. J. Am. Chem. Soc. 1984, 106, 3374–3376. 44 Inokuchi, T.; Matsumoto, S.; Nishiyama, T.; Torii, S. J. Org. Chem. 1990, 55, 462–466. 45 (a) Anelli, P.L.; Banfi, S.; Montanari, F.; Quici, S. J. Org. Chem. 1989, 54, 2970–2972 (b) Siedlecka, R.; Skarzewski, J.; Mlochowski, J. Tetrahedron Lett. 1990, 31, 2177–2180 (b) Davis, N.J.; Flitsch, S.L. Tetrahedron Lett. 1993, 34, 1181–1184. 46 (a) Spyroudis, S.; Varvoglis, A. Synthesis 1975, 445–447 (b) Narasaka, K.; Morikawa, A.; Saigo, K.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 1977, 50, 2773–2776 (c) Muller, P.; Godoy, J. Tetrahedron Lett. 1981, 22, 2361–2364 (d) Muller, P.; Godoy, J.; Helv. Chim. Acta 1983, 66, 1790–1795 (d) Togo, H.; Aoki, M.; Kuramochi, T.; Yokoyama, M. J. Chem. Soc., Perkin Trans. 1 1993, 2417–2427 (e) Magnus, P.; Lacour, J.; Evans, P.A.; Roe, M.B.; Hulme, C. J. Am. Chem. Soc. 1996, 118, 3406–3418. 47 See for the use of [bis(acetoxy)iodo]benzene (BAIB): Epp, J.B.; Widlanski, T.S. J. Org. Chem. 1999, 64, 293–295. 48 De Luca, L.; Giacomelli, G.; Masala, S.; Porcheddu, A. J. Org. Chem. 2003, 68, 4999–5001. Unpredictable outcomes were reported by Chambers et al. (see ref 22b). 49 Bragd, P.L.; Van Bekkum, H.; Besemer, A.C. Topics in Catalysis, 2004, 27, 49–66. 50 Despite extensive studies, the exact mechanism is still unclear: (a) Semmelhack, M.F.; Schmid, C.R.; Cortés, D.A. Tetrahedron Lett. 1986, 27, 1119–1122 (b) Ma, Z.; Bobbitt, J.M. J. Org. Chem. 1991, 56, 6110–6114 (c) De Nooy, A.E.J.; Besemer, A.C.; Van Bekkum, H. Tetrahedron 1995, 51, 8023–8032.

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General Introduction    51 Haller, M.; Boons, G.-J. J. Chem. Soc., Perkin Trans. 1 2001, 814–822. 52 Garegg, P.J.; Oscarson, S.; Tedebark, U. J. Carbohydr. Chem. 1998, 17, 587–594. 53 Chauvin, A.-L.; Nepogodiev, S.A.; Field, R.A. J. Org. Chem. 2005, 70, 960–966. 54 Yeung, B.K.S.; Hill, D.C.; Janicka, M.; Petillo, P.A. Org. Lett. 2000, 2, 1279–1282. 55 Litjens, R.E.J.N.; Den Heeten, R.; Timmer, M.S.M.; Overkleeft, H.S.; Van der Marel, G.A. Chem. Eur. J. 2005, 11, 1010–1016. 56 Huang, L.; Teumelsan, N.; Huang, X. Chem. Eur. J. 2006, 12, 5246–5252. 57 (a) Chiba, T.; Sinaÿ, P. Carbohydr. Res. 1986, 151, 379–389 (b) Chiba, T.; Jacquinet, J.-C.; Sinaÿ, P.; Petitou, M.; Choay, J. Carbohydr. Res. 1988, 174, 253–264 (c) Schmidt, R.R.; Rücker, E. Tetrahedron Lett. 1980, 21, 1421–1424. 58 Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1988, 173, 306–315. 59 (a) Jacquinet, J.-C.; Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Torri, G.; Sinaÿ, P. Carbohydr. Res. 1984, 130, 221–241 (b) Sinaÿ, P.; Jacquinet, J.-C.; Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Torri, G. Carbohydr. Res. 1984, 132, C5–C9 (c) Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Jacquinet, J.-C.; Sinaÿ, P.; Torri, G. Carbohydr. Res. 1987, 167, 67–75. 60 (a) Schmidt, R.R.; Grundler, G. Synthesis 1981, 885–887 (b) Marra, A.; Dong, X.; Petitou, M.; Sinaÿ, P. Carbohydr. Res. 1989, 195, 39–50 (c) Hada, N.; Ogino, T.; Yamada, H.; Takeda, T. Carbohydr. Res. 2001, 334, 7–17. 61 Tabeur, C.; Machetto, F.; Mallet, J.-M.; Duchaussoy, P.; Petitou, M.; Sinaÿ, P. Carbohydr. Res. 1996, 281, 253–276. 62 Maruyama, M.; Takeda, T.; Shimizu, N.; Hada, N.; Yamada, H. Carbohydr. Res. 2000, 325, 83–92. 63 Kochetkov, N.K. Tetrahedron 1987, 43, 2389–2436. 64 Vogel, C.; Steffan, W.; Ott, A.Y.; Betanelli, V.I. Carbohydr. Res. 1992, 237, 115–129. 65 Steffan, W.; Vogel, C.; Kristen, H. Carbohydr. Res. 1990, 204, 109–120. 66 Partial epimerization around the anomeric centre of the acceptor occurred, which is earlier observed using this glycosylation method: Kitov, P.I.; Tsvetkov, Y.E.; Bachinowski, L.V.; Kochetkov, N.K. Bioorg. Khim. 1989, 15, 1416–1422. 67 Nilsson, M.; Svahn, C.M.; Westman, J. Carbohydr. Res. 1993, 246, 161–172. 68 Throughout the text, glycosides having the ido-configuration are depicted in the 1C4 conformation. For more details see Codée, J.D.C. Thesis Leiden University 2004. 69 This is a modified version of the Corey-conditions: Corey, E.J.; Samuelsson, B. J. Org. Chem. 1984, 49, 4735–4735. 70 The authors mentioned that no acid scavenger was added to the reaction mixture. DMTST-mediated glycosylations are usually performed without adding base, even in the presence of acid labile protective groups. Original article: Fügedi, P.; Garegg, P. Carbohydr. Res. 1986, 149, C9–C12. 71 Magaud, D.; Grandjean, C.; Doutheau, A.; Anker, D.; Shevchik, V.; Cotte-Pattat, N.; Robert-Baudouy, J. Carbohydr. Res.; 2000, 314, 189–199. 72 Initially it was established that coupling between two D-galacturonic acid esters could not be effected using bromide, trichloroacetimidate and n-pentenyl functionalized donors. 73 Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Sinaÿ, P.; Jacquinet, J.-C.; Torri, G. Carbohydr. Res. 1986, 147, 221–236 74 Pews-Davtyan, A.; Pirojan, A.; Shaljyan, I.; Awetissjan, A.A.; Reinke, H.; Vogel, C. J. Carbohydr. Chem. 2003, 22, 939–962. See also: (a) Lönn, H.; Lönngren, J.; Thompson, J.L. Carbohydr. Res. 1984, 132, 39–44 (b) Oscarson, S. Topics in Current Chemistry, Thiem, J. ed., Springer Verlag, Berlin, 1997, 172–201.

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Chapter 1    75 Elimination is also found in the degradation of uronic acid containing polysaccharides: Lindberg, B.; Lönngren, J.; Thompson, J.L. Carbohydr. Res. 1973, 28, 351–357. 76 Magaud, D.; Grandjean, C.; Doutheau, A.; Anker, D.; Shevchik, V.; Cotte-Pattat, N.; Robert-Baudouy, J. Tetrahedron Lett. 1997, 38, 241–244. 77 Konradsson, P.; Udodong, U.E.; Fraser-Reid, B. Tetrahedron Lett. 1990, 31, 4313–4316. 78 Better results were obtained using sonicated Jones oxidation: Allanson, N.M.; Liu, D; Chi, F.; Jain, R.K.; Chen, A.; Ghosh, M.; Hong, L.; Sofia, M.J. Tetrahedron Lett. 1998, 39, 1889–1892. 79 The authors also tried the rather unknown tris(4-bromophenyl) ammoniumyl hexachloroantimonate without success: Marra, A.; Mallet, J.-M.; Amatore, C.; Sinaÿ, P. Synlett 1990, 572–574. 80 Garegg, P.J., Olsson, L.; Oscarson, S. J. Org. Chem. 1995, 60, 2200–2204. 81 (a) Nakano, T.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1990, 31, 1597–1600 (b) Goto, F.; Ogawa, T. Tetrahedron Lett. 1992, 33, 5099–5102. 82 Pfitzner, K.E.; Moffat, J.G. J. Am. Chem. Soc. 1965, 87, 5661–5670. 83 Oscarson, S.; Svahnberg, P. J. Chem. Soc., Perkin Trans. 1 2001, 873–879. 84 Misra, A.K.; Roy, N. Carbohydr. Res. 1995, 278, 103–111. 85 Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168. 86 Van den Bos, L.J.; Litjens, R.E.J.N.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2005, 7, 2007–2010. 87 (a) Crich, D.; Sun, S. J. Org. Chem. 1997, 62, 1198–1199 (b) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020 (c) Crich, D.; Li, W.; Li, H. J. Am. Chem. Soc. 2004, 126, 15081–15086. 88 (a) Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279 (b) Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522. 89 Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819–6825. 90 Van den Bos, L.J.; Dinkelaar, J.; Overkleeft, H.S.; Van der Marel, G.A. J. Am. Chem. Soc. 2006, 128, 13066–13067. 91 (a) Crich, D.; Sun, S.; Brunckova, J. J. Org. Chem. 1996, 61, 605–615. (b) Crich, D.; Sun, S. J. Org. Chem. 1996, 61, 4506–4507 92 Jensen, H.H.; Nordstrøm, L.U.; Bols, M. J. Am. Chem. Soc. 2004, 126, 9205–9213. 93 (a) Iwahara, S.; Suemori, N.; Ramli, N.; Takegawa, K. Biosci. Biotech. Biochem. 1995, 59, 1082–1085 (b) Jikibara, T.; Takegawa, K.; Iwahara, S. J. Biochem. 1992, 111, 225–229. 94 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326. 95 Codée, J.D.C.; Stubba, B.; Schiattarella, M.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. J. Am. Chem. Soc. 2005, 127, 3767–3773. 96 Van den Bos, L.J.; Duivenvoorden, B.A.; De Koning, M.C.; Filippov, D.V.; Overkleeft, H.S.; Van der Marel, G.A. Eur. J. Org. Chem. 2007, 116–124.

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Chapter 2 │  Thioglycuronides: Synthesis and    Application in the Assembly of    Acidic Oligosaccharides

Abstract: Partially protected thioglycuronic acids are prepared efficiently by chemo- and regioselective oxidation of the corresponding thioglycosides using the TEMPO/BAIB reagent combination. After esterification, the thioglycuronic acids proved to be useful as both donor and acceptor in sulfonium-mediated condensations towards acidic di- and trisaccharides.1

Introduction Uronic acids are present in a wide array of biologically relevant oligosaccharides, polysaccharides and glycoconjugates.2 As a result, flexible and straightforward synthesis routes towards these important molecules should have major impact on research in glycobiology. Although it is well established that thioglycosides are versatile synthons en route towards such carbohydrate targets,3 approaches utilizing thioglycuronic acids are scarce. This can be explained by the lack of efficient synthetic protocols for the preparation of suitably protected thioglycuronides.4 In addition, thioglycuronic acids have been shown to be rather poor glycosyl donors, generally requiring the presence of activating protective groups. As part of a program directed towards the development of efficient methodologies for the preparation of biologically relevant oligosaccharides,5 Codée et al. recently reported a novel sequential glycosylation strategy (Figure 1, route A).6 The method is based on Ph2SO (6b)/Tf2O-mediated condensation7 (I) of 1-hydroxyl donor 1 and partially protected

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Chapter 2   

thioglycoside 2 to afford the corresponding thiodisaccharide. In the next glycosylation event (II) successive treatment of the intermediate thio function with sulfonium triflate species 7a or 7b, generated in situ from BSP (6a)/Tf2O8 or Ph2SO (6b)/Tf2O9 (see insert in Figure 1), followed by addition of a suitably protected nucleophile 3 afforded the desired trisaccharide. The potency of the novel activator systems 7a and 7b in these syntheses encouraged us to use the highly unreactive thioglycuronides in the aforementioned glycosylation sequence. This clearly called for an efficient mode of synthesis to access a wide variety of thioglycuronic acid synthons.

Figure 1. A OP (PO)n

O 1

I OH

HO

S

sulfonium activator system

OP O

HO

R = Et or Ph P = protecting group

B

O

orthogonal glycosylation

(OP)n SR 2

II

chemo- and OH regioselective O oxidation O HO

OP

(OP)n SR 4

(OP)n SR 5

O

HO

OTf R

6

a: R = piperidino (BSP) b: R = phenyl (Ph2SO) O

trisaccharides

N

TEMPO

R OTf

-60°C

O (OP)n OP 3

S

Tf2O

7

OAc I

OAc

BAIB

This Chapter describes the 2,2,6,6-tetramethyl-1-piperidinyloxyl free radical (TEMPO)/[bis(acetoxy)iodo]benzene (BAIB)-mediated chemo- and regioselective oxidation of readily available partially protected thioglycosides 4 as a powerful means to obtain the corresponding thioglycuronic acids (Figure 1, route B).10,11 After esterification of the carboxylate functions, these partially-protected thioglycuronides 5 can be incorporated in the chemoselective glycosylation strategy, in the same way as key building block 2, to furnish acidic oligosaccharides.

Results and Discussion Piancatelli and coworkers recently reported the oxidation of primary alcohols into their corresponding aldehydes using catalytic TEMPO and an equimolar amount of BAIB as cooxidant.10 Interestingly, this oxidation protocol could also be applied to alcohols containing sulfo- and selenoethers. In this Chapter, application of this reagent combination for the selective oxidation of suitably protected thioglycosides to the corresponding thioglycuronic acids is established. As the initial research objective, the compatibility of the TEMPO/BAIB system with thioglucopyranosides 8 and 9 was evaluated (Scheme 1). Treatment of ethyl

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Thioglycuronides: Synthesis and Application    12

2,3,4-tri-O-benzyl-1-thio-β-D-glucopyranoside (8) with a catalytic amount of TEMPO and excess BAIB in a mixture of dichloromethane (DCM) and water (2:1), followed by methylation of carboxylate 10 using freshly prepared diazomethane, afforded thioglucuronide 12 in a rewarding yield of 88% over the two steps. It should be noted that careful monitoring of the reaction mixture by TLC and timely quenching with an aqueous thiosulfate solution adequately suppresses unwanted over-oxidation to the sulfoxide and/or sulfone. Following the same sequence of reactions, ethyl 2,3-di-O-benzyl-1-thio-β-D-glucopyranoside (9)12 was converted into methyl ester 13 in 85% yield (based on 9), demonstrating the excellent chemoand regioselective nature of this method.

Scheme 1. OH RO BnO

O OBn 8: R = Bn 9: R = H

a SEt

RO BnO

COOH O OBn 10: R = Bn 11: R = H

b SEt

RO BnO

COOMe O

SEt

OBn 12: R = Bn 13: R = H

Reagents and conditions: (a) TEMPO, BAIB, DCM, H2O, 10: 88%, 11: 92% (b) CH2N2, DMF, 12: quant., 13: 92%.

The results of the TEMPO/BAIB oxidation of a variety of thioglycosides and one selenoglycoside are summarized in Table 1. Phenyl 2,3,4-tri-O-benzoyl-1-seleno-α-Dgalactopyranoside (14) was readily transformed into methyl galacturonate 16, via acid 15 (entry 1). In the same way, subjection of isopropylidene-protected mannoside 17 and benzylated galactoside 2013 to the two-step procedure produced esters 19 and 22 in good yield (entry 2 and 3). Entries 4-10 further demonstrate the selectivity of this method for the oxidation of primary alcohols in the presence of both a thio-functionality and a secondary alcohol. Both S-phenyl- and S-ethylthioglycosides can be employed, as illustrated by the equally efficient transformation of 2314 and 2415 into 26 and 28, respectively. Furthermore, both the starting glycoside (glucose, glucosamine, galactose, and idose) and the nature of the protective groups (benzyl, benzoyl, isopropylidene, tert-butyldimethylsilyl, azide, and phthalimide) can be readily varied without major implications on the outcome of the oxidation step (all yields are within the range of 60-90%). As the next research objective, the applicability of the obtained thioglycuronates in sulfonium triflate-mediated glycosylation reactions was established. The reactivity of these thioglycuronic acids towards various activating systems is considerably reduced compared to the corresponding thioglycosides due to the electron withdrawing effect of the carboxyl function. It was reasoned that the sulfonium triflate activators 7a and 7b would be sufficiently electrophilic for reaction with the deactivated anomeric thio-function. Indeed, treatment of

33   

33

Chapter 2    Table 1.

entry

substrate BzO

product BzO

OH O

1

BzO

ROOC O O BnO O

SPh 17

BnO

OH O

3

BnO

SPh

7

O

35

SPh

8

O

HO BzO

SR

OBz 23: R = Et 24: R = Ph

HO BzO

SR

OBz 25: R1=Et, R2=H (76%)a,c 1 2 26: R =Et, R =Me (83%)b,c

SEt

N3

SPh

HO BzO

COOR O

SEt

38 OH

HO

BnO

SPh

COOR O

BnO

SPh

OBz 42: R = H (85%)a,c 43: R = Me (93%)b,c

OBz

9

COOR O

BnO

N3 39: R = H (88%)a,c 40: R = Me (80%)b,c

O 1

SPh

OBn 36: R = H (85%)a,c 37: R = Me (93%)b,c

OBn

HO

COOR2 O

SPh

COOR O

NPhth 33: R = H (67%)a,c 34: R = Me (80%)b,c

HO

OH

BnO

21: R = Ha 22: R = Me (60%)b,c,d

OH

HO TBSO

OH

OBn

20

4

COOR O

BnO

OBn

HO BzO

32

O

SPh 18: R = H (quant.)a,c 19: R = Me (81%)b,c

SPh

NPhth

HO

O

BnO

6

SePh a,c

OH O O

product

O

HO TBSO

1 5: R = H (61 %) b,c 1 6: R = Me (8 3%)

14

2

BzO

SePh

substrate OH

COOR O

B zO

BzO

BnO

entry

41

27: R1=Ph, R2=H (81%)a,c 28: R1=Ph, R2=Me (86%)b,c

OH

5

O

HO BnO

OBn 29

a

SPh

HO BnO

COOR O

HO

SPh

OBn a

30: R = H 31: R = Me (71%)b,c,d

10

OBn O OH

SEt

OBz 44

ROOC

OBn O

SEt

OH OBz 45: R = H (84%)a,c 46: R = Me (96%)b,c

TEMPO, BAIB, DCM/H2O (2/1). b CH2N2, DMF. c isolated yields. d over 2 reaction steps.

donor 47 with 7b showed nice activation of the donor glycoside. Compared to the standard conditions reported by the Crich laboratory, higher activation temperatures and longer activation times were needed.16 Addition of primary alcohol acceptor 4817 to the reaction mixture afforded disaccharide 49 as an anomeric mixture (α/β = 1/3) in 71% yield (Table 2., entry 1). Decrease in the reactivity of the acceptor nucleophile, as in 50, leads to an increase in the formation of the α-product (entry 2). This trend was further pronounced when galacturonic acid ester 52 was subjected to Ph2SO/Tf2O-mediated glycosylation with acceptors 48 and 50 to give disaccharides 53 and 54 in good yields (entry 3 and 4). The BSP/Tf2O reagent combination is equally efficient in the activation and glycosidation of donor 52 with deactivated acceptor 55, despite the slightly lower reactivity of BSP due to its stabilizing nitrogen lone pair (entry 5).9,18 Even the highly “disarmed”, fully acylated donor 57 was smoothly activated by the BSP/Tf2O activator system affording disaccharide 58 in good yield (entry 6). These results compare favorably with the results published by the group 34   

34

Thioglycuronides: Synthesis and Application   

of Oscarson who had to install at least two activating benzyl functions or a 3,4tetraisopropyldisiloxyl group at the thioglucuronate donors to obtain sufficient activation with dimethyl(methylthio)sulfonium triflate (DMTST).4,19

Table 2.

entry

1

RO BnO

donor

acceptor

COOMe O

OH

SPh

OBn

O

BnO BnO

activator yield (α/β)b,c Ph2SO/Tf2O 71% (1/3)

disaccharide COOMe O

AcO BnO

BnO

BnO OMe 48

31: R=H 47: R=Ac (77%)a,b Ph O

2

47

O

OpMP

Ph2SO/Tf2O 63% (1/1)

MeOOC AcO BnO

BnO

Ph2SO/Tf2O 80% (1/2)

48

SPh

O

OBn

OpMP

O OBn

51

AcO

3

O O

O BnO

OBn 50

COOMe O

BnO OMe

Ph O

HO

RO

O

O BnO BnO 49

COOMe O

BnO BnO

37: R=H 52: R=Ac (quant.)a,b

O

O BnO BnO 53

BnO OMe

Ph AcO

4

52

Ph2SO/Tf2O 68% (1/0)

50

BnO

CO2Me O O O BnO

O 54

BnO

5

COOMe O

HO

52

OBn 55

6

RO BzO

COOMe O

SPh

OBz 28: R=H 57: R=Ac (quant.)a,b a

48

AcO

OpMP

BSP/Tf2O 57% (1/0)

BSP/Tf2O 68% (0/1)

BnO

AcO BzO

OpMP

O OBn

COOMe O OBn COOMe O BnO OpMP O OBn 56

COOMe O

O

O

OMe OBn

OBz 58

BnO

OBn

Ac2O, pyridine. b isolated yields. c anomeric ratios were determined by 1H NMR spectroscopy.

35   

35

Chapter 2   

Since the results in Table 2. demonstrate that 1-thioglycuronates can be readily applied as donors in the sulfonium activator-mediated disaccharide synthesis, attention was focused on the implementation of 1-thioglucuronic acid ester 13 in the sequential chemoselective synthesis of trisaccharide 62. This protected carbohydrate corresponds to the (→2)-α-DGlcpNAc-(1→4)-α-D-GlcpA-α-(1→2)-β-D-Galf-(1→) trisaccharide present in the capsular polysaccharide of the Fungus Fusarium sp. M7-1.20 Activation of hemiacetal donor 5921 with diphenylsulfonium bistriflate 7b followed by treatment with uronic acid acceptor 13 gave the α-linked thiodisaccharide 60 as the sole product in a gratifying yield of 67% (Scheme 3). Tritert-butylpyrimidine (TTBP) was used as acid scavenger.22 In the second glycosylation event, 60 was smoothly activated under the agency of BSP/Tf2O and condensed with partially protected galactono-1,4-lactone 6123 to give the desired trisaccharide 62 as an anomeric mixture (α/β = 4/1) in 35% yield. Despite several attempts, the yield for this reaction could not be optimized further.

Scheme 3. OAc O

BnO BnO

N3

+

HO BnO

OH

OAc

COOMe O

a SEt

N3 O BnO

OBn

59

13

60

OAc BnO BnO

O

BnO BnO

62

SEt

OBn

O

O N3 O BnO

COOMe O

BnO

O

O

COOMe BzO O

H O

O

b

O

H

O

O

BzO 61 OH

O

Reagents and conditions: (a) 59, Ph2SO, Tf2O, TTBP, DCM, -60°C then 13, 67% (b) 60, BSP, Tf2O, TTBP, DCM, -60°C then 61, 35% (α/β = 4/1).

Conclusion This Chapter presents a novel and efficient strategy for the regio- and chemoselective oxidation of thioglycosides to the corresponding thioglycuronides. In turn, these thioglycuronides proved to be useful as both acceptors and donors in the synthesis of acidic oligosaccharides as demonstrated by the assembly of trisaccharide 62. Further, the electrophilic Ph2SO/Tf2O and BSP/Tf2O activator systems proved capable in activating even highly deactivated 1-thioglycuronides. This approach nicely complements contemporary synthetic efforts towards uronic acid containing oligosaccharides, which are often based on the introduction of the carboxylate functions after oligosaccharide assembly. The efficiency of oligosaccharide synthesis using thioglycuronic acid donors is somewhat compromized by 36   

36

Thioglycuronides: Synthesis and Application   

their reduced reactivity compared to the parent thioglycosides. The yields observed in the examples presented here are in the range of 35–80%. However, the partial loss in glycosylation efficiency is more than compensated by the reduced number of protective group manipulations required.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Jeol JNM-FX-200 (200/50.1 MHz), a Bruker AV-400 (400/100 MHz) and a Brüker DMX-600 (600/150 MHz) spectrometer. Chemical shifts are given in ppm (δ) relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and QStar Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Melting points were measured on a Büchi Schmelzpunktbestimmungsapparat nach dr. Tottoli (+ pat 320.388) and were uncorrected. Traces of water in the donor and acceptor glycosides, Ph2SO, BSP and TTBP were removed by co-evaporation with toluene. BSP8 and TTBP22 were synthesised as described by Crich et al. Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled immediately prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography and TLC were of technical grade and distilled before use. Flash chromatography was performed on Baker silica gel (0.063 – 0.200 mm). TLC-analysis was conducted on DC-fertigfolien (Schleicher & Schuell, F1500, LS254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in ethanol followed by charring at ~150°C. General Procedure for the TEMPO/BAIB-Mediated Oxidation: To a vigorously stirred solution of 0.3 mmol thioglycoside in 1 mL DCM and 0.5 mL H2O was added 0.06 mmol TEMPO (0.2 equiv.) and 0.75 mmol BAIB (2.5 equiv.). Stirring was allowed until TLC indicated complete conversion of the starting material to a lower running spot (~45 min). The reaction mixture was quenched by the addition of 10 mL Na2S2O3 solution (10% in H2O) and 10 mL NaHCO3 (sat., aq.). The mixture was then extracted twice with EtOAc (10 mL) and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash column chromatography using EtOAc/petroleum ether/AcOH afforded the pure glycuronic acids. N.B.: NMR-spectra of the various unprotected carboxylic acids sometimes showed low intensity of the C-5 signals. In all cases, the intensity of the peak was restored after methylation of the carboxylate group. General Procedure for the Esterification using Diazomethane: A solution of 0.2 mmol thio uronic acid in 1 mL DMF was treated with a freshly prepared ethereal solution of diazomethane24 (~0.03 M) until the evolution of gas ceased [CAUTION: diazomethane is highly explosive]. The reaction mixture was then treated with 1 mL AcOH and concentrated in vacuo. Flash column chromatography using EtOAc/petroleum ether afforded the methyl uronates.

37   

37

Chapter 2    General Procedure for Glycosylations using BSP/Tf2O or Ph2SO/Tf2O: A solution of donor (1 equiv), benzenesulfinylpiperidine (1.3 equiv.) or diphenyl sulfoxide (1.3 equiv.) and tri-tert-butylpyrimidine (2.5 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30 min. The mixture was cooled to -60°C before triflic acid anhydride (1.3 equiv.) was added. The mixture was allowed to warm to -40°C in 15min followed by addition of acceptor (1.5 equiv.) in DCM (0.15M). Stirring was continued and the reaction mixture was allowed to warm to 0°C. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding coupled disaccharides. Ethyl 2,3,4-tri-O-benzyl-1-thio-β-D-glucopyranosiduronic acid (10): TLC: 50% EtOAc/PE (5% AcOH); MP: 125°C; [α]D22: -22° (c = 1, CHCl3); IR (neat, cm-1): 1074, 1454, 1726; 1H OBn NMR (400 MHz, MeOD) δ = 1.25 (t, 3H, J = 7.4 Hz, CH3 SEt), 2.62 – 2.75 (m, 2H, CH2 SEt), 3.39 (t, 1H, J = 9.2 Hz, H-2), 3.67 (t, 1H, J = 8.7 Hz, H-3), 3.75 (t, 1H, J = 9.2 Hz, H-4), 3.93 (d, 1H, J = BnO BnO

COOH O

SEt

9.6 Hz, H-5), 4.59 (d, 1H, J = 10.0 Hz, H-1), 4.62 – 4.86 (m, 6H, 3xCH2Ph), 7.22 – 7.31 (m, 15H, H Arom); 13C NMR (100 MHz, MeOD) δ = 15.6 (CH3 SEt), 25.8 (CH2 SEt), 75.9 (CH2 Bn), 76.2 (CH2 Bn), 76.7 (CH2 Bn), 79.0 (C-5), 81.0 (C-4), 82.5 (C-2), 86.6 (C-1), 86.7 (C-3), 128.6 – 129.3 (CH Arom), 139.3 (Cq Bn), 139.5 (Cq Bn), 139.8 (Cq Bn), 172.0 (C=O COOH); HRMS: [M+NH4]+ calcd for C29H36O6SN 509.19924, found 509.19894.

HO BnO

COOH O

SEt

OBn

Ethyl 2,3-di-O-benzyl-1-thio-β-D-glucopyranosiduronic acid (11): TLC: 50% EtOAc/PE (5% AcOH); [α]D22: -24° (c = 1, CHCl3); IR (neat, cm-1): 1085, 1251, 1712, 1776, 3386; 1H NMR (400 MHz, MeOD) δ = 1.23 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.59 – 2.74 (m, 2H, CH2

SEt), 3.51 (t, 1H, J = 9.4 Hz, H-2), 3.51 (t, 1H, J = 8.8 Hz, H-3), 3.75 (t, 1H, J = 9.2 Hz, H-4), 3.81 (d, 1H, J = 9.6 Hz, H-5), 4.52 (d, 1H, J = 9.6 Hz, H-1), 4.62 (d, 1H, J = 10.4 Hz, CHHPh), 4.75 (d, 1H, J = 11.2 Hz, CHHPh), 4.78 (d, 1H, J = 10.8 Hz, CHHPh), 4.90 (d, 1H, J = 10.8 Hz, CHHPh), 7.06 – 7.31 (m, 10H, H Arom); 13 C NMR (100 MHz, MeOD) δ = 15.5 (CH3 SEt), 25.6 (CH2 SEt), 73.3 (C-5), 76.2 (CH2 Bn), 76.5 (CH2 Bn), 80.0 (C-4), 82.0 (C-2), 86.4 (C-1), 87.0 (C-3), 128.5 – 129.9 (CH Arom), 139.4 (Cq Bn), 140.0 (Cq Bn), 172.2 (C=O COOMe); HRMS: [M+NH4]+ calcd for C22H30O6SN 436.1788, found 436.1793.

BnO BnO

COOMe O

SEt

OBn

Methyl (ethyl 2,3,4-tri-O-benzyl-1-thio-β-D-glucopyranoside) uronate (12): TLC: 25% EtOAc/PE; MP: 125°C; [α]D22: -10° (c = 1, CHCl3); IR (neat, cm-1): 1026, 1074, 1284, 1745; 1H NMR (400 MHz, CDCl3) δ = 1.30 (t, 3H, CH3 SEt), 2.75 (m, 2H, CH2 SEt), 3.47

(t, 1H, J = 9.2 Hz, H-2), 3.69 (t, 1H, J = 8.8 Hz, H-3), 3.75 (t, 1H, J = 9.2 Hz, H-4), 3.89 (d, 1H, J = 9.6 Hz, H5), 4.50 (d, 1H, J = 9.6 Hz, H-1), 4.61 (d, 1H, J = 10.8 Hz, CHHPh), 4.73 (d, 1H, J = 10.4 Hz, CHHPh), 4.77 (d, 1H, J = 10.8 Hz, CHHPh), 4.84 (d, 1H, J = 10.8 Hz, CHHPh), 4.90 (d, 1H, J = 11.2 Hz, CHHPh), 4.92 (d, 1H, J = 10.0 Hz, CHHPh), 7.29 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 14.9 (CH3 SEt), 25.0 (CH2 SEt), 52.3 (CH3 COOMe), 75.0 (CH2 Bn), 75.4 (CH2 Bn), 76.0 (CH2 Bn), 78.1 (C-5), 81.2 (C-4), 81.2 (C-2), 85.8 (C1 and C-3), 127.8 – 128.4 (CH Arom), 137.8 (Cq Bn), 138.2 (Cq Bn), 168.7 (C=O COOMe); HRMS: [M+NH4]+ calcd for C30H38O6SN 540.2414, found 540.2422.

HO BnO

COOMe O

Methyl (ethyl 2,3-di-O-benzyl-1-thio-β-D-glucopyranoside) uronate (13): TLC 50%

EtOAc/PE; [α]D22: -30° (c = 1, CHCl3); IR (neat, cm-1): 1026, 1062, 1174, 1209, 1357, 1438, 1454, 1496, 1747, 2329, 2866; 1H NMR (400 MHz, CDCl3) δ = 1.32 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.68 – 2.34 (m, 2H, CH2 SEt), 2.89 (bs, 1H, OH), 3.41 (t, 1H, J = 9.2 Hz, H-2), 3.56 (t, 1H, J = 8.6 Hz, HOBn

38   

38

SEt

Thioglycuronides: Synthesis and Application    3), 3.80 (s, 3H, CH3 COOMe), 3.83 (s, 1H, H-5), 3.90 (t, 1H, J = 9.4 Hz, H-4), 4.52 (d, 1H, J = 9.6 Hz, H-1), 4.75 (d, 1H, J = 10.0 Hz, CHHPh), 4.88 (s, 2H, CH2 Bn), 4.89 (d, 1H, J = 11.2 Hz, CHHPh), 7.13 – 7.35 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 15.0 (CH3 SEt), 25.2 (CH2 SEt), 52.7 (CH3 COOMe), 71.9 (C4), 75.5 (CH2 Bn), 76.2 (CH2 Bn), 77.6 (C-5), 80.6 (C-2), 85.1 (C-3), 85.9 (C-1), 126.6 – 128.8 (CH Arom), 137.8 (Cq Bn), 138.4 (Cq Bn), 169.6 (C=O COOMe); HRMS: [M+NH4]+ calcd for C23H28O6S 450.1945, found 450.1930.

BzO

Phenyl 2,3,4-tri-O-benzoyl-1-seleno-α-D-galactopyranosiduronic acid (15): TLC: 50% EtOAc/PE (5% AcOH); IR (neat, cm-1): 1026, 1068, 1091, 1178, 1211, 1257, 1315, 1450,

COOH O

BzO

1583, 1600, 1724; 1H NMR (400 MHz, MeOD) δ = 4.89 (s, 1H, H-5), 5.44 (d, 1H, J = 10.0 Hz, H-1), 5.66 (t, 1H, J = 9.8 Hz, H-2), 5.74 (dd, 1H, J = 9.6 Hz, J = 3.2 Hz, H-3), 6.21 (d, 1H, J = 2.4 Hz, H-4), 7.08 – 7.96 (m, 20H, H Arom); 13C NMR (100 MHz, MeOD) δ = 69.8 (C-2), 71.1 (C-4), 74.0 (C3), 77.5 (C-5), 80.9 (C-1), 127.2 (Cq SePh), 129.1 – 137.7 (CH Arom), 130.2 (Cq Bz), 130.5 (Cq Bz), 130.6 (Cq Bz), 166.6 (C=O Bz or COOH), 169.5 (C=O Bz or COOH); HRMS: [M+NH4]+ calcd for C33H30O9SeN BzO

SePh

664.1080, found 664.1098. BzO

Methyl (phenyl 2,3,4-tri-O-benzoyl-1-seleno-α-D-galactopyranoside) uronate (16): TLC: 30% EtOAc/PE; IR (neat, cm-1): 1001, 1024, 1066, 1091, 1178, 1211, 1257, 1438, 1450, 1600,

COOMe O

BzO

1726; 1H NMR (400 MHz, CDCl3) δ = 4.63 (s, 1H, H-5), 5.20 (d, 1H, J = 9.6 Hz, H-1), 5.58 (dd, 1H, J = 9.6 Hz, J = 3.2 Hz, H-3), 5.73 (t, 1H, J = 9.8 Hz, H-2), 6.18 (dd, 1H, J = 2.8 Hz, J = 0.8 Hz, H-4), 7.20 – 7.99 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.6 (CH3 COOMe), 68.0 (C3), 69.2 (C-4), 72.4 (C-2), 76.8 (C-5), 80.2 (C-1), 125.7 (Cq SePh), 128.2 – 136.7 (CH Arom), 128.8 (Cq Bz), 164.9 (C=O Bz or COOMe), 165.4 (C=O Bz or COOMe), 166.1 (C=O Bz or COOMe); HRMS: [M+NH4]+ calcd BzO

SePh

for C34H32O9SeN 678.1237, found 678.1235. HOOC BnO O

O

O SPh

Phenyl 4-O-benzyl-2,3-O-isopropylidene-1-thio-α-D-mannopyraniduronic acid (18): TLC: 50% EtOAc/PE (5% AcOH); [α]D22: +104° (c = 1, CHCl3); 1H NMR (400 MHz, MeOD) δ = 1.33 (s, 3H, CH3 isoprop), 1.42 (s, 3H, CH3 isoprop), 3.97 (dd, 1H, J = 7.4 Hz, J = 6.0 Hz, J =

5.6 Hz, H-4), 4.26 (dd, 1H, J = 5.4 Hz, J = 3.6 Hz, H-2), 4.32 (t, 1H, J = 5.6 Hz, H-3), 4.59 (d, 1H, J = 7.2 Hz, H-5), 4.67 (d, 1H, J = 11.6 Hz, CHHPh), 4.75 (d, 1H, J = 11.6 Hz, CHHPh), 5.68 (d, 1H, J = 3.2 Hz, H-1), 7.07 – 7.55 (m, 10H, H Arom); 13C NMR (100 MHz, MeOD) δ = 26.5 (CH3 isoprop), 28.1 (CH3 isoprop), 71.9 (C-5), 73.7 (CH2 Bn), 76.1 (C-2), 77.2 (C-4), 77.9 (C-3), 84.7 (C-1), 111.0 (Cq isoprop), 128.8 – 133.0 (CH Arom), 134.3 (Cq SPh), 139.1 (Cq Bn), 172.3 (C=O); HRMS: [M+NH4]+ calcd for C22H28O6SN 434.1632, found 434.1695. MeOOC BnO O

O

O SPh

Methyl (phenyl 4-O-benzyl-2,3-O-isopropylidene-1-thio-α-D-mannopyranoside) uronate (19): TLC: 30% EtOAc/PE; [α]D 22: +43° (c = 0.6, CHCl3); IR (neat, cm-1): 1043, 1093, 1234, 1735; 1 H NMR (400 MHz, CDCl3) δ = 1.29 (s, 3H, CH3 isoprop), 1.42 (s, 3H, CH3 isoprop), 3.63 (s,

3H, CH3 COOMe), 3.90 (dd, 1H, J = 7.8 Hz, J = 6.0 Hz, H-4), 4.22 (dd, 1H, J = 5.6 Hz, J = 3.6 Hz, H-2), 4.27 (t, 1H, J = 5.8 Hz, H-3), 4.56 (d, 1H, J = 8.4 Hz, H-5), 4.59 (d, 1H, J = 12.4 Hz, CHHPh), 4.72 (d, 1H, J = 11.2 Hz, CHHPh), 5.60 (d, 1H, J = 3.6 Hz, H-1), 7.17 – 7.47 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 26.1 (CH3 isoprop), 27.7 (CH3 isoprop), 52.3 (CH3 COOMe), 70.7 (C-5), 72.9 (CH2 Bn), 75.0 (C-2), 76.0 (C-4), 76.7 (C-3), 83.7 (C-1), 110.0 (Cq isoprop), 127.6 – 131.9 (CH Arom), 132.8 (Cq SPh), 137.5 (Cq Bn), 169.4 (C=O); HRMS: [M+NH4]+ calcd for C23H30O6SN 448.1788, found 448.1833.

39   

39

Chapter 2    BnO

COOH O

SPh

BnO

Phenyl 2,3,4-tri-O-benzyl-1-thio-β-D-galactopyranosiduronic acid (21): Experimental data were in full accord with reported literature data.25

OBn

BnO

COOMe O

BnO

Methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-β-D-galactopyranoside) uronate (22): Experimental SPh

OBn

COOH O

data were in full accord with literature.25 ESI: 588.2 [M+NH4]+; HRMS: [M+NH4]+ calcd for C34H38O6SN 588.24144, found 588.24011. Ethyl 2,3-di-O-benzoyl-1-thio-β-D-glucopyranosiduronic acid (25): TLC: 50% EtOAc/PE

(5% AcOH); IR (neat, cm-1): 1072, 1272, 1450, 1604, 1720; 1H NMR (400 MHz, MeOD) δ = 1.22 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.67 – 2.77 (m, 2H, CH2 SEt), 4.07 (t, 1H, J = 9.4 Hz, H-4), 4.13 (d, 1H, J = 10.0 Hz, H-5), 4.93 (d, 1H, J = 9.2 Hz, H-1), 5.35 (t, 1H, J = 9.8 Hz, H-2), 5.62 (t, 1H, J = 9.2 Hz, H-3), 7.09 – 7.92 (m, 10H, H Arom); 13C NMR (100 MHz, MeOD) δ = 15.3 (CH3 SEt), 25.0 (CH2 SEt), 71.0 (C-4), 71.8 (C-2), 77.4 (C-3), 80.1 (C-5), 84.7 (C-1), 128.9 – 134.3 (CH Arom), 129.7 (Cq Bz), 130.2 (Cq HO BzO

SEt

OBz

Bz), 166.5 (C=O Bz or COOH), 167.2 (C=O Bz or COOH); HRMS: [M+H]+ calcd for C22H23O8S 469.09276, found 469.09314.

HO BzO

COOMe O

SEt

Methyl (ethyl 2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside) uronate (26): TLC: 30% EtOAc/PE; [α]D22: +63° (c = 1, CHCl3); IR (neat, cm-1): 1066, 1272, 1724, 3471; 1H NMR

(400 MHz, CDCl3): δ = 1.20 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.72 (m, 2H, CH2 SEt), 3.82 (s, 3H, CH3 COOMe), 4.14 (d, 1H, J = 10.0 Hz, H-5), 4.21 (t, 1H, J = 9.2 Hz, H-4), 4.80 (d, 1H, J = 10.0 Hz, H-1), 5.46 (t, 1H, J = 9.8 Hz, H-2), 5.62 (t, 1H, J = 9.2 Hz, H-3), 7.28 – 7.96 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 14.6 (CH3 SEt), 24.3 (CH2 SEt), 52.7 (CH3 COOMe), 69.8 (C-2), 70.3 (C-4), 76.0 (C-3), 78.3 (C-5), 84.2 (C-1), 128.2 – 133.2 (CH Arom), 129.0 (Cq Bz), 165.2 (C=O Bz or COOMe), 166.3 (C=O Bz or COOMe), OBz

168.7 (C=O Bz or COOMe); HRMS: [M+NH4]+ calcd for C23H28O8SN 483.10841, found 483.10828.

HO BzO

COOH O OBz

SPh

Phenyl 2,3-di-O-benzoyl-1-thio-α-D-glucopyranosiduronic acid (27): TLC: EtOAc (2% AcOH); [α]D22: +61° (c = 1; CHCl3); IR (neat, cm-1): 707, 1066, 1271, 1712; 1H NMR (400 MHz, MeOD): δ = 4.12 (t, 1H, J = 9.4 Hz, H-4), 5.26 (d,1H, J = 10.0 Hz, H-5), 5.29 (t, 1H,

J = 10.0 Hz, H-1), 5.37 (t, 1H, J = 9.4 Hz, H-2), 5.74 (t, 1H, J = 9.2 Hz, H-3), 7.21 – 8.06 (m, 15H, H Arom); 13 C NMR (100 MHz, MeOD): δ = 70.1 (C-4), 70.9 (C-2), 76.6 (C-3), 78.9 (C-5), 86.1 (C-1), 128.1 – 133.5 (CH Arom), 129.2 (Cq Bz), 132.2 (Cq SPh), 165.5 (C=O Bz), 166.3 (C=O Bz), 170.4 (C=O COOH); HRMS: [M+H]+ calcd for C26H23O8S 469.09276, found 469.09314. Methyl (phenyl 2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside) uronate (28): TLC: 30% EtOAc/PE; [α]D22: +63° (c = 1, CHCl3); IR (neat, cm-1): 707, 1066, 1272, 1724, 3471; 1H SPh OBz NMR (400 MHz, CDCl3) δ = 3.52 (bs, 1H, OH), 3.87 (s, 3H, CH3 COOMe), 4.13 (d, 1H, J = 9.6 Hz, H-5), 4.18 (t, 1H, J = 9.2 Hz, H-4), 4.99 (d, 1H, J = 10.0 Hz, H-1), 5.41 (t, 1H, J = 9.8 Hz, H-2), 5.58 HO BzO

COOMe O

(t, 1H, J = 9.0 Hz, H-3), 7.26 – 8.02 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 53.0 (CH3 COOMe), 69.7 (C-4), 70.3 (C-2), 76.2 (C-3), 78.0 (C-5), 86.9 (C-1), 128.4 – 133.4 (CH Arom), 128.9 (Cq Bz), 129.0 (Cq Bz), 131.9 (Cq SPh), 165.0 (C=O Bz or COOMe), 166.5 (C=O Bz or COOMe), 168.8 (C=O Bz or COOMe); HRMS: [M+H]+ calcd for C27H25O8S 517.0928, found 517.0933.

40   

40

Thioglycuronides: Synthesis and Application    Methyl (phenyl 2,3-di-O-benzyl-1-thio-β-D-glucopyranoside) uronate (31): TLC: 25% EtOAc/PE; [α]D22: -54° (c = 1.8, CHCl3); IR (neat, cm-1): 694, 734, 979, 1026, 1141, 1203, OBn 1355, 1436, 1759; 1H NMR (600 MHz, CDCl3) δ = 2.95 (bs, 1H, OH-4), 3.40 (dd, 1H, J = 9.9 Hz, J = 9.0 Hz, J = 8.4 Hz, H-2), 3.51 (t, 1H, J = 9.0 Hz, J = 8.4 Hz, H-3), 3.73 (s, 3H, CH3 COOMe), 3.76 COOMe O

HO BnO

SPh

(d, 1H, J = 9.6 Hz, H-5), 3.82 (t, 1H, J = 9.6 Hz, J = 9.0 Hz, H-4), 4.61 (d, 1H, J = 9.6 Hz, H-1), 4.67 (d, 1H, J = 10.2 Hz, CHHPh), 4.78 (s, 2H, CH2 Bn), 4.80 (d, 1H, J = 10.2 Hz, CHHPh), 7.16 – 7.48 (m, 15H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 52.7 (CH3 COOMe), 71.7 (C-4), 75.5 (CH2 Bn), 75.6 (CH2 Bn), 77.7 (C-5), 79.6 (C-2), 85.1 (C-3), 88.3 (C-1), 127.1 – 132.6 (CH Arom), 133.1 (Cq SPh), 137.7 (Cq Bn), 138.2 (Cq Bn), 169.5 (C=O COOMe); ESI-MS: 503.1 [M+Na]+; HRMS: [M+Na]+ calcd for C27H28O6SNa 503.14988, found 503.14941. COOH O

HO TBSO

SPh

NPhth

Phenyl 3-O-tert-butyldimethylsilyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranosiduronic acid (33): TLC: EtOAc (5% AcOH); [α]D22: +39.6° (c = 1, CHCl3); IR (neat, cm-1): 1085, 1251, 1386, 1593, 1712, 1776, 3386; 1H NMR (400 MHz, CDCl3) δ = -0.19 (s, 3H, CH3

TBDMS), 0.14 (s, 3H, CH3 TBDMS), 0.66 (s, 9H, tBu TBDMS), 3.65 (t, 1H, J = 9.2 Hz, H-4), 3.93 (d, 1H, J = 10.0 Hz, H-5), 4.26 (t, 1H, J = 10.2 Hz, H-2), 4.49 (t, 1H, J = 9.2 Hz, H-3), 5.65 (d, 1H, J = 10.4 Hz, H-1), 7.22 – 7.93 (m, 9H, H Arom); 13C NMR (100 MHz, CDCl3) δ = -5.0 (CH3 TBDMS), -3.4 (CH3 TBDMS), 18.7 (Cq tBu TBDMS), 26.2 (tBu TBDMS), 57.6 (C-2), 74.8 (C-4), 75.1 (C-3), 81.0 (C-5), 85.8 (C-1), 124.3 – 135.8 (CH Arom), 132.9 (Cq SPh), 134.2 (Cq NPhth), 169.0 (C=O NPhth), 169.7 (C=O NPhth), 177.2 (C=O COOH); HRMS: [M+NH4]+ calcd for C26H35O7N2SSi 547.1929, found 547.1987.

HO TBSO

COOMe O

SPh

Methyl (phenyl 3-O-tert-butyldimethylsilyl-2-deoxy-2-phtalimido-1-thio-β-D-glucopyranoside) uronate 34: : TLC: 50% EtOAc/PE (5% AcOH); [α]D22: +93.2° (c = 1, CHCl3); IR

(neat, cm-1): 1110, 1384, 1710; 1H NMR (400 MHz, CDCl3) δ = -0.30 (s, 3H, CH3 TBDMS), 0.02 (s, 3H, CH3 TBDMS), 0.61 (s, 9H, tBu TBDMS), 3.10 (bs, 1H, OH), 3.76 (dd, 1H, J = 11.0 Hz, J = 8.4 Hz, J = 5.6 Hz, H-4), 3.81 (s, 3H, CH3 COOMe), 4.02 (d, 1H, J = 10.0 Hz, H-5), 4.24 (t, 1H, J = 10.2 Hz, H-2), 4.43 (dd, 1H, J = 9.6 Hz, J = 8.4 Hz, H-3), 5.55 (d, 1H, J = 10.8 Hz, H-1), 7.13 – 7.84 (m, 10H, H Arom); 13 C NMR (100 MHz, CDCl3) δ = -5.3 (CH3 TBDMS), -4.1 (CH3 TBDMS), 17.8 (Cq tBu TBDMS), 25.5 (tBu NPhth

TBDMS), 52.8 (CH3 COOMe), 55.6 (C-2), 72.9 (C-3), 73.1 (C-4), 77.7 (C-5), 84.5 (C-1), 123.2 – 134.2 (CH Arom), 131.7 (Cq SPh), 132.1 (Cq Phth), 169.7 (C=O NPhth or COOMe); ESI-MS: 566.2 [M+Na]+. HO

COOH O

BnO

SPh

Phenyl 2,3-di-O-benzyl-1-thio-β-D-galactopyranosiduronic acid (36): TLC: 50% EtOAc/PE (5% AcOH); [α]D22: -11° (c = 0.8, CHCl3); IR (neat, cm-1): 1027, 1086, 1725; 1H NMR (400

MHz, CDCl3) δ = 3.62 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz, H-3), 3.74 (t, 1H, J = 9.4 Hz, H-2), 4.06 (s, 3H, H-5), 4.43 (bs, 1H, H-4), 4.64 (d, 1H, J = 9.6 Hz, H-1), 4.65 (d, 1H, J = 11.6 Hz, CHHPh), 4.73 (d, 1H, J = 11.2 Hz, CHHPh), 4.75 (d, 1H, J = 10.0 Hz, CHHPh), 4.82 (d, 1H, J = 10.4 Hz, CHHPh), 5.57 (bs, 1H, COOH), 7.12 – 7.58 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 67.4 (C-4), 72.2 (CH2 Bn), 75.8 (CH2 Bn), 76.2 (C-2), 76.6 (C-5), 81.5 (C-3), 87.8 (C-1), 127.9 – 132.6 (CH Arom), 132.9 (Cq SPh), 137.2 (Cq Bn), OBn

137.9 (Cq Bn), 169.9 (C=O COOH); HRMS: [M+Na]+ calcd for C26H26O6SNa 467.15229, found 467.15396 HO BnO

COOMe O

SPh

Methyl (phenyl 2,3-di-O-benzyl-1-thio-β-D-galactopyranoside) uronate (37): TLC: 50% EtOAc/PE; [α]D22: -8° (c = 0.6, CHCl3); IR (neat, cm-1): 1026, 1083, 1716; 1H NMR (400

MHz, CDCl3) δ = 2.49 (s, 1H, OH), 3.63 (dd, 1H, J = 8.8 Hz, J = 3.2 Hz, H-3), 3.77 (t, 1H, J OBn = 9.0 Hz, H-2), 3.84 (s, 3H, CH3 COOMe), 4.07 (s, 1H, H-5), 4.39 (s, 1H, H-4), 4.59 (d, 1H, J = 10.0 Hz, H-1), 4.68 (d, 1H, J = 11.2 Hz, CHHPh), 4.73 (d, 2H, J = 10.0 Hz, CHHPh, CHHPh), 4.83 (d, 1H, J = 10.4 Hz,

41   

41

Chapter 2    CHHPh), 7.13 – 8.01 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.7 (CH3 COOMe), 67.8 (C-4), 72.2 (CH2 Bn), 75.8 (CH2 Bn), 76.3 (C-2), 76.7 (C-5), 81.7 (C-3), 87.8 (C-1), 127.8 – 132.8 (CH Arom), 133.1 (Cq SPh), 137.2 (Cq Bn), 138.0 (Cq Bn), 168.2 (C=O); HRMS: [M+H]+ calcd for C27H29O6S 503.14972, found 503.14988. Ethyl 2-azido-3-O-benzoyl-2-deoxy-1-thio-β-D-glucopyranosiduronic acid (39): TLC: 50% EtOAc/PE (5% AcOH); [α]D22: -1° (c = 1, CHCl3); IR (neat, cm-1): 1026, 1068, 1116, 1261, N3 1421, 1716, 2108; 1H NMR (400 MHz, MeOD) δ = 1.31 (t, 3H, J = 7.0 Hz, CH3 SEt), 2.79 (m, 2H, CH2 SEt), 3.67 (t, 1H, J = 10.0 Hz, H-2), 3.91 (t, 1H, J = 9.4 Hz, H-4), 3.98 (d, 1H, J = 9.6 Hz, H-5), HO BzO

COOH O

SEt

4.69 (d, 1H, J = 10.4 Hz, H-1), 5.25 (t, 1H, J = 9.4 Hz, H-3), 7.47 – 8.11 (m, 5H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 15.5 (CH3 SEt), 25.5 (CH2 SEt), 65.1 (C-2), 71.2 (C-4), 77.9 (C-3), 80.1 (C-5), 85.7 (C-1), 129.6 – 134.5 (CH Arom), 167.3 (C=O Bz), 172.0 (C=O COOH); HRMS: [M+H]+ calcd for C15H18N3O6S 368.09108, found 368.09232. Methyl (ethyl 2-azido-3-O-benzoyl-2-deoxy-1-thio-β-D-glucopyranoside) uronate (40): TLC: 30% EtOAc/PE; [α]D22: -2° (c = 0.8, CHCl3); IR (neat, cm-1): 1091, 1211, 1265, 1450, N3 1732, 2110; 1H NMR (400 MHz, CDCl3) δ = 1.36 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.82 (m, 2H. CH2 SEt), 3.62 (t, 1H, J = 10.0 Hz, H-2), 3.82 (s, 3H, CH3 COOMe), 4.00 (d, 1H, J = 9.6 Hz, H-5), 4.05 (t, 1H, J HO BzO

COOMe O

SEt

= 9.4 Hz, H-4), 4.55 (d, 1H, J = 10.0 Hz, H-1), 5.26 (t, 1H, J = 9.6 Hz, J = 9.2 Hz, H-3), 7.45 – 8.10 (m, 5H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 14.9 (CH3 SEt), 25.2 (CH2 SEt), 52.9 (CH3 COOMe), 63.6 (C-2), 70.2 (C-4), 76.7 (C-3), 78.1 (C-5), 85.2 (C-1), 128.5 – 133.6 (CH Arom), 129.1 (Cq Bz), 166.4 (C=O Bz or COOMe), 168.7 (C=O Bz or COOMe); ESI-MS: 404.1 [M+Na]+. Phenyl 2-O-benzoyl-3-O-benzyl-1-thio-β-D-galactopyranosiduronic acid (42): TLC: 50% EtOAc/PE (5% AcOH); [α]D22: +53° (c = 1, CHCl3); IR (neat, cm-1): 1026, 1068, 1091, BnO OBz 1263, 1431, 1600, 1720; 1H NMR (400 MHz, MeOD) δ = 3.80 (d, 1H, J = 9.6 Hz, H-3), 4.21 (s, 1H, H-5), 4.53 (d, 1H, J = 12.4 Hz, CHHPh), 4.54 (s, 1H, H-4), 4.64 (d, 1H, J = 12.4 Hz, CHHPh), 4.90 (d, 1H, J = 8.4 Hz, H-1), 5.49 (t, 1H, J = 9.8 Hz, H-2), 7.00 – 7.93 (m, 15H, H Arom); 13C NMR (100 MHz, MeOD) HO

COOH O

SPh

δ = 67.0 (C-4), 69.7 (C-2), 70.5 (CH2 Bn), 78.1 (C-5), 79.4 (C-3), 86.6 (C-1), 127.1 – 133.0 (CH Arom), 129.8 (Cq SPh), 133.9 (Cq Bz), 137.8 (Cq Bn), 165.6 (C=O Bz or COOH); ESI-MS: 503.3 [M+Na]+. HO

COOMe O

BnO

SPh

Methyl (phenyl 2-O-benzoyl-3-O-benzyl-1-thio-β-D-galactopyranoside) uronate (43): TLC: 50% EtOAc/PE; [α]D22: +29° (c = 1, CHCl3); IR (neat, cm-1): 1213, 1265, 1662, 1726, 1760;

H NMR (400 MHz, CDCl3) δ = 3.74 (dd, 1H, J = 9.2 Hz, J = 2.8 Hz, H-3), 3.84 (s, 3H, CH3 COOMe), 4.18 (s, 1H, H-5), 4.50 (d, 1H, J = 12.0 Hz, CHHPh), 4.51 (s, 1H, H-4), 4.67 (d, 1H, J = 12.4 Hz, CHHPh), 4.74 (d, 1H, J = 10.4 Hz, H-1), 5.52 (t, 1H, J = 9.2 Hz, H-2), 7.02 – 8.02 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.6 (CH3 COOMe), 67.3 (C-4), 68.9 (C-2), 71.2 (CH2 Bn), 77.0 (C-5), 78.3 (C-3), 86.6 (C-1), 127.8 – 133.1 (CH Arom), 129.6 (Cq SPh), 132.4 (Cq Bz), 136.6 (Cq Bn), 165.1 (C=O Bz or COOMe), OBz

1

167.9 (C=O Bz or COOMe); HRMS: [M+H]+ calcd for C27H27O7S 512.1738, found 512.1797.

HOOC

OBn O

SEt

Ethyl 2-O-benzoyl-3-O-benzyl-1-thio-α/β-L-idopyranosiduronic acid (45): TLC: 50% EtOAc/PE (5% AcOH); %); IR (neat, cm-1) 1066, 1716; α-anomer: 1H NMR (400 MHz,

OH OBz MeOD): δ = 1.27 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.68 (m, 2H, CH2 SEt), 3.87 (s, 1H, H-3), 4.14 (m, 1H, H-4), 4.68 (d, 1H, J = 12.0 Hz, CHHPh), 4.80 (d, 1H, J = 12.0 Hz, CHHPh), 5.08 (s, 1H, H-5), 5.26 (s, 1H, H-2), 5.52 (s, 1H, H-1), 7.12 – 8.17 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 15.5 (CH3

42   

42

Thioglycuronides: Synthesis and Application    SEt), 27.3 (CH2 SEt), 69.0 (C-4), 70.2 (C-5), 71.0 (C-2), 73.4 (CH2 Bn), 76.8 (C-3), 84.0 (C-1), 128.7 – 134.4 (CH Arom), 130.8 (Cq Bz), 138.9 (Cq Bn), 166.9 (C=O Bz or COOMe), 173.2 (C=O Bz or COOMe); β-anomer: 1 H NMR (400 MHz, CDCl3): δ = 1.30 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.78 (m, 2H, CH2 SEt), 3.82 (s, 1H, H-3), 4.08 (m, 1H, H-4), 4.57 (s, 1H, H-5), 4.76 (d, 1H, J = 12.0 Hz, CHHPh), 4.81 (d, 1H, J = 12.0 Hz, CHHPh), 5.21 (s, 1H, H-1), 5.22 (s, 1H, H-2), 7.12 – 8.17 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 15.5 (CH3 SEt), 26.4 (CH2 SEt), 67.8 (C-4), 71.3 (C-2), 73.4 (CH2 Bn), 76.4 (C-3), 77.2 (C-5), 83.0 (C-1), 128.7 – 134.4 (CH Arom), 130.7 (Cq Bz) 139.0 (Cq Bn), 167.3 (C=O Bz or COOMe), 172.4 (C=O Bz or COOMe); HRMS: [M+NH4]+ calcd for C22H28O7SN 450.1581, found 450.1568. Methyl (ethyl 2-O-benzoyl-3-O-benzyl-1-thio-α/β-L-idopyranoside) uronate (46): TLC: 50% EtOAc/PE; IR (neat, cm-1): 1091, 1720, 1760; α-anomer: 1H NMR (400 MHz, CDCl3) OH OBz δ = 1.30 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.66 (m, 2H, CH2 SEt), 2.93 (bd, 1H, J = 11.2 Hz, OH), 3.79 (s, 3H, CH3 COOMe), 3.88 (s, 1H, H-3), 4.10 (m, 1H, H-4), 4.63 (d, 1H, J = 12.0 Hz, CHHPh), 4.83 (d, 1H, J = 12.0 Hz, CHHPh), 5.28 (s, 1H, H-5), 5.32 (s, 1H, H-2), 5.56 (s, 1H, H-1), 7.12 – 8.00 (m, 10H, H MeOOC

SEt

OBn O

Arom); 13C NMR (100 MHz, CDCl3): δ = 14.8 (CH3 SEt), 26.7 (CH2 SEt), 52.1 (CH3 COOMe), 68.1 (C-4), 68.3 (C-5), 69.5 (C-2), 72.0 (CH2 Bn), 73.6 (C-3), 83.0 (C-1), 127.5 – 133.4 (CH Arom), 128.8 (Cq Bz), 136.9 (Cq Bn), 164.7 (C=O Bz or COOMe), 169.6 (C=O Bz or COOMe); β-anomer: 1H NMR (400 MHz, CDCl3): δ = 1.30 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.75 (m, 2H, CH2 SEt), 2.87 (bd, 1H, J = 11.6 Hz, OH), 3.79 (s, 3H, CH3 COOMe), 3.99 (s, 1H, H-3), 4.06 (m, 1H, H-4), 4.60 (s, 1H, H-5), 4.72 (d, 1H, J = 12.0 Hz, CHHPh), 4.80 (d, 1H, J = 11.6 Hz, CHHPh), 5.15 (s, 1H, H-1), 5.28 (s, 1H, H-2), 7.12 – 8.00 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 14.7 (CH3 SEt), 25.6 (CH2 SEt), 52.1 (CH3 COOMe), 67.3 (C-4), 69.9 (C-2), 72.4 (CH2 Bn), 74.2 (C-3), 76.0 (C-5), 81.9 (C-1), 127.5 – 133.4 (CH Arom), 128.7 (Cq Bz), 136.9 (Cq Bn), 165.1 (C=O Bz or COOMe), 168.7 (C=O Bz or COOMe); HRMS: [M+NH4]+ calcd for C23H30O7SN 464.1737, found 464.1739. Methyl (phenyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-β-D-galactopyranoside) uronate (47): TLC: 25% EtOAc/PE; [α]D22: -60° (c = 2.5, CHCl3); IR (neat, cm-1): 694, 734, 979, 1026, OBn 1141, 1203, 1355, 1436, 1759; 1H NMR (600 MHz, CDCl3) δ = 1.85 (s, 3H, CH3 Ac), 3.46 (dd, 1H, J = 9.6 Hz, J = 9.0 Hz, H-2), 3.62 (t, 1H, J = 9.6 Hz, J = 9.0 Hz, H-3), 3.66 (s, 3H, CH3 COOMe), 3.82 AcO BnO

COOMe O

SPh

(d, 1H, J = 9.6 Hz, H-5), 4.57 (d, 1H, J = 9.6 Hz, H-1), 4.60 (d, 1H, J = 10.2 Hz, CHHPh), 4.62 (d, 1H, J = 10.2 Hz, CHHPh), 4.72 (d, 1H, J = 10.2 Hz, CHHPh), 4.78 (d, 1H, J = 10.2 Hz, CHHPh), 5.06 (t, 1H, J = 9.6 Hz, H4), 7.15 – 7.50 (m, 15H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 20.6 (CH3 Ac), 52.6 (CH3 COOMe), 70.8 (C-4), 72.9 (C-5), 75.5 (CH2 Bn), 76.2 (C-2), 83.1 (C-3), 87.6 (C-1) 127.4 – 132.6 (CH Arom), 132.5 (Cq SPh), 137.6 (Cq Bn), 137.8 (Cq Bn), 167.5 (C=O Ac or COOMe), 139.5 (C=O Ac or COOMe); ESI-MS: 545.2 [M+Na]+; HRMS: [M+Na]+ calcd for C29H30O7SNa 545.16045, found 545.16022.

AcO BnO

COOMe O BnO

O BnO BnO

O BnO OMe

Methyl 2,3,4-tri-O-benzyl-6-O-(methyl (4-acetyl-2,3-di-O-benzyl-α/β-D-glucopyranoside) uronate)-α-D-glucopyranoside (49): Donor 47 was glycosylated with acceptor 4817 in the same way as described in the general procedure using

Ph2SO/Tf2O delivering the title compound 49 as a colorless oil. TLC: 25% EtOAc/PE; IR (neat, cm-1): 694, 734, 979, 1026, 1141, 1203, 1355, 1436, 1759; β-anomer: 1H NMR (400 MHz, CDCl3) δ = 1.91 (s, 3H, CH3 Ac), 3.31 (s, 3H, CH3 OMe), 3.45 (t, 1H, J= 10.0 Hz, H-4), 3.49 (dd, 1H, J = 10.0 Hz, J = 3.5 Hz, H-2), 3.54 (dd, 1H, J = 9.5 Hz, J = 7.5 Hz, H-2’), 3.58 – 3.62 (m, 2H, H-6, H-3’), 3.74 (s, 3H, CH3 COOMe), 3.79 (d, 1H, J = 10.0 Hz, H-5’), 3.80 (m, 1H, H-5), 3.99 (t, 1H, J = 9.5 Hz, H-3), 4.13 (d, 1H, J = 11.0 Hz, H-6), 4.48 – 4.98 (m, 10H, 5xCH2Ph), 4.36 (d, 1H, J = 7.5 Hz, H-1’), 4.58 (d, 1H, J = 3.5 Hz, H-1), 5.09 (t, 1H, J = 9.5 Hz, H-4’), 7.18 – 7.35 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.6 (CH3 Ac),

43   

43

Chapter 2    52.6 (CH3 COOMe), 55.1 (CH3 OMe), 68.6 (C-6), 70.2 (C-5’), 71.0 (C-4’), 72.7 (C-5), 73.3 (CH2 Bn), 74.8 (CH2 Bn), 75.0 (CH2 Bn), 75.1 (CH2 Bn), 75.6 (CH2 Bn), 77.9 (C-4), 79.8 (C-2), 81.1 (C-2’ or C-3’), 81.2 (C-3’ or C-2’), 81.8 (C-3), 97.9 (C-1), 103.6 (C-1’), 127.5 – 128.4 (CH Arom), 137.9 (2xCq Bn), 138.0 (Cq Bn), 138.2 (Cq Bn), 138.7 (Cq Bn), 167.8 (C=O Ac or COOMe), 169.6 (C=O Ac or COOMe); ESI: 894.5 [M+NH4]+; HRMS: [M+NH4]+ calcd for C51H60O13N 894.32297, found 894.33388. para-Methoxyphenyl 2-O-benzyl-4,6-O-benzylidene-3-O-(methyl 4-O-acetyl2,3-di-O-benzyl-α/β-D-glucopyranosyluronate)-β-D-galactopyranoside (51): Donor 47 was glycosylated with acceptor 50 in the same way as described in

Ph MeOOC AcO BnO

O

O

O O

the general procedure using Ph2SO/Tf2O delivering the title compound 51 as a colorless oil. TLC: 25% EtOAc/PE; IR (neat, cm-1): 694, 734, 979, 1026, 1141, 1203, 1355, 1436, 1759; α-anomer: 1H NMR (400 MHz, CDCl3) δ = 1.87 (s, 3H, CH3 Ac), 3.51 – 3.53 (m, 1H, H-5), 3.63 – 3.65 (m, 4H, H-2, CH3 pMP), 3.76 (s, 3H, CH3 COOMe), 4.05 (dd, 1H, J = 7.6 Hz, J = 2.4 Hz, H3), 4.05 (t, 1H, J = 7.6 Hz, H-3’), 4.10 (d, 1H, J = 10.0 Hz, H-6), 4.17 (t, 1H, J = 7.6 Hz, H-2), 4.32 – 4.39 (m, BnO

O

OpMP

OBn

2H, H-4, H-6), 4.49 (d, 1H, J = 8.4 Hz, H-5’), 4.61 – 5.19 (m, 6H, 3xCH2Ph), 4.92 (d, 1H, J = 7.6 Hz, H-1), 5.01 (t, 1H, J = 7.6 Hz, H-4’), 5.63 (s, 1H, CHPh), 6.81 (d, 2H, J = 9.0 Hz, 2xCH pMP), 7.03 (d, 2H, J = 9.0 Hz, 2xCH pMP), 7.07 – 7.61 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.5 (CH3 Ac), 52.5 (CH3 pMP), 55.6 (CH3 COOMe), 66.7 (C-5), 68.2 (C-5’), 69.0 (C-6), 74.5 (C-4’), 74.9 (CH2 Bn), 75.1 (CH2 Bn), 75.3 (CH2 Bn), 75.5 (C-4), 75.9 (C-3), 77.3 (C-3’), 78.0 (C-2’), 79.0 (C-2), 92.4 (C-1’), 100.7 (CHPh), 103.2 (C-1), 114.4 (CH pMP), 119.6 (CH pMP), 126.2 – 129.7 (CH Arom), 137.5 (Cq Bn), 137.9 (Cq Bn), 138.2 (Cq Bn), 151.4 (Cq pMP), 155.3 (Cq pMP), 168.1 (C=O COOMe or Ac), 169.7 (C=O Ac or COOMe); ESI: 894.5 [M+NH4]+; HRMS: [M+NH4]+ calcd for C50H56O14N 894.36953, found 894.36334. Methyl (phenyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-β-D-galactopyranoside) uronate (52): A solution of 734 mg compound 37 (1.53 mmol) in 5 mL pyridine and 2 mL Ac2O was stirred OBn for 3h before 5 mL MeOH was added. The reaction mixture was concentrated under reduced pressure and several times co-evaporated with toluene. Flash column chromatography using EtOAc/petroleum ether afforded 798 mg of the title compound 52 (1.53 mmol, quant) as a colorless oil. TLC: 25% EtOAc/PE; AcO

COOMe O

BnO

SPh

[α]D22: -32° (c = 1, CHCl3); IR (neat, cm-1): 1230, 1749; 1H NMR (400 MHz, CDCl3) δ = 2.11 (s, 3H, CH3 Ac), 3.67 (m, 2H, H-2, H-5), 3.78 (s, 3H, CH3 COOMe), 4.52 (d, 1H, J = 11.2 Hz, CHHPh), 4.63 (d, 1H, J = 9.6 Hz, H-1), 4.74 (d, 1H, J = 10.4 Hz, CHHPh), 4.75 (d, 1H, J = 10.0 Hz, CHHPh), 4.77 (d, 1H, J = 10.0 Hz, CHHPh), 5.82 (d, 1H, J = 1.2 Hz, H-4), 7.25 – 7.40 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 52.6 (CH3 COOMe), 67.6 (C-4), 72.0 (CH2 Bn), 75.6 (C-3), 76.1 (CH2 Bn), 76.7 (C-2 or C-5), 80.6 (C-5 or C-2), 87.8 (C-1), 127.8 – 128.8 (CH Arom), 137.2 (Cq Bn), 138.0 (Cq Bn), 167.0 (C=O Ac or COOMe), 169.9 (C=O Ac or COOMe); HRMS: [M+H]+ calcd for C29H31O7S 523.17850, found 523.18024 AcO

Methyl 2,3,4-tri-O-benzyl-6-O-(methyl 4-O-acetyl-2,3-di-O-benzyl-α/β-Dgalacto-pyranosyluronate)-α-D-glucopyranoside (53): Donor 52 was glycosylated

COOMe O

BnO

O

with acceptor 4817 in the same way as described in the general procedure using Ph2SO/Tf2O delivering the title compound 53 as a colorless oil. TLC: 25% EtOAc/PE; IR (neat, cm-1): 694, 734, 979, 1026, 1141, 1203, 1355, 1436, 1759; β-anomer: 1H NMR (400 MHz, CDCl3) δ = 2.08 (s, 3H, CH3 Ac), 3.29 (s, 3H, CH3 OMe), 3.48 (m, 2H, H-2, H-4), 3.55 (dd, 1H, J = 9.6 Hz, J = BnO

O BnO BnO

BnO OMe

3.6 Hz, H-3’), 3.64 – 3.70 (m, 2H, H-5 and H-2’), 3.74 (s, 3H, CH3 COOMe), 3.79 – 3.85 (m, 1H, H-6), 4.00 (m, 2H, H-3, H-5’), 4.23 (d, 1H, J = 9.6 Hz, H-6), 4.29 (d, 1H, J = 8.0 Hz, H-1’), 4.48 – 4.98 (m, 10H, 5xCH2Ph), 4.58 (d, 1H, J = 3.2 Hz, H-1), 5.75 (d, 1H, J = 2.4 Hz, H-4’), 7.17 – 7.49 (m, 25H, H Arom); 13C NMR (100

44   

44

Thioglycuronides: Synthesis and Application    MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.4 (CH3 COOMe), 54.9 (CH3 OMe), 67.0 (C-4’), 68.8 (C-6), 69.9 (C-5), 72.0 (CH2 Bn), 72.4 (C-5’), 73.2 (CH2 Bn), 74.7 (CH2 Bn), 75.6 (CH2 Bn), 75.7 (CH2 Bn), 77.9 (C-2 or C-4), 78.0 (C-2’), 78.6 (C-3’), 79.8 (C-4 or C-2), 81.9 (C-3), 97.9 (C-1), 103.7 (C-1’), 127.4 – 128.4 (CH Arom), 137.5 (Cq Bn), 138.0 (Cq Bn), 138.4 (Cq Bn), 138.7 (Cq Bn), 138.8 (Cq Bn), 167.2 (C=O Ac or COOMe), 169.9 (C=O Ac or COOMe); ESI: 899.6 [M+Na]+; HRMS: [M+Na]+ calcd for C51H56O13Na 899.36131, found 899.35809. para-Methoxyphenyl 2-O-benzyl-4,6-O-benzylidene-3-O-(methyl 4-O-acetyl-2,3di-O-benzyl-α-D-galactopyranosyluronate)-β-D-galactopyranoside (54): Donor 52

Ph AcO

CO2Me O O O

was glycosylated with acceptor 50 in the same way as described in the general procedure using Ph2SO/Tf2O delivering the title compound 54 as a colorless oil. OBn TLC: 25% EtOAc/PE; IR (neat, cm-1): 694, 734, 979, 1026, 1141, 1203, 1355, 1 1436, 1759; H NMR (400 MHz, CDCl3) δ = 2.02 (s, 3H, CH3 Ac), 3.47 (s, 1H, H-5), 3.58 (s, 3H, CH3 OMe), 3.76 (s, 3H, CH3 COOMe), 3.83 (dd, 1H, J = 9.6 Hz, J = 3.2 Hz, H-2’), 3.86 (dd, 1H, J = 10.0 Hz, J = 3.6 Hz, HBnO

BnO O

O

OpMP

3), 3.95 (dd, 1H, J = 10.0 Hz, J = 3.32 Hz, H-3’), 4.08 (d, 1H, J = 11.2 Hz, H-6), 4.13 (dd, 1H, J = 10.0 Hz, J = 8.0 Hz, H-2), 4.33 (d, 1H, J = 3.2 Hz, H-4), 4.36 (d, 1H, J = 11.6 Hz, H-6), 4.50 (d, 1H, J = 11.2 Hz, CHHPh), 4.54 (d, 1H, J = 12.0 Hz, CHHPh), 4.63 (d, 1H, J = 11.6 Hz, CHHPh), 4.70 (d, 2H, J = 10.8 Hz, 2xCHHPh), 4.75 (d, 1H, J = 1.2 Hz, H-5’), 4.90 (d, 1H, J = 8.0 Hz, H-1), 5.06 (d, 1H, J = 10.8 Hz, CHHPh), 5.24 (d, 1H, J = 3.6 Hz, H-1’), 5.52 (d, 1H, J = 2.0 Hz, H-4’), 5.57 (s, 1H, CHPh), 6.82 (d, 2H, J = 9.0 Hz, 2xCH pMP), 7.03 (d, 2H, J = 9.0 Hz, 2xCH pMP), 7.07 – 7.28 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.2 (CH3 OMe), 55.6 (CH3 COOMe), 66.3 (C-5), 68.9 (C-4’), 68.8 (C-5’), 69.2 (C-6), 71.4 (C-4), 72.2 (CH2 Bn), 72.6 (CH2 Bn), 74.0 (C-3), 74.4 (C-2’), 75.0 (C-3’), 75.4 (CH2 Bn), 76.8 (C-2), 93.4 (C-1’), 101.3 (CHPh), 103.2 (C-1), 107.4 (CH Arom pMP), 119.6 (CH Arom pMP), 123.8 – 128.9 (CH Arom), 137.5 (Cq Bn), 137.9 (Cq Bn), 138.2 (Cq Bn), 138.2 (Cq Bn), 151.4 (Cq pMP), 155.3 (Cq pMP), 168.1 (C=O COOMe or Ac), 169.7 (C=O Ac or COOMe); ESI: 899.6 [M+Na]+; HRMS: [M+Na]+ calcd for C50H52O14Na 899.32493, found 899.31935. AcO

COOMe O OBn COOMe BnO O BnO OpMP O OBn

Methyl (4-methoxyphenyl 2,4-di-O-benzyl-3-O-(methyl 4-O-acetyl-2,3-di-O-ben-

zyl-α-D-galactopyranosyluronate)-β-D-galactopyranoside) uronate (56): Donor 52 was glycosylated with acceptor 55 in the same way as described in the general procedure using BSP/Tf2O delivering 51 mg (0.056 mmol, 57%) of the title compound 56 as a colorless oil. TLC: 20% EtOAc/toluene; [α]D22: +68° (c = 1, CHCl3); IR (neat, cm-1): 1026, 1089, 1215, 1506, 1747; 1H NMR (400 MHz, CDCl3) δ = 2.01 (s, 3H, CH3 Ac), 3.58 (s, 3H, CH3 COOMe), 3.67 (s, 3H, CH3 COOMe), 3.76 (s, 3H, CH3 pMP), 3.89 (m, 2H, H-3, H-2’), 3.96 (dd, 1H, J = 10.0 Hz, J = 3.2 Hz, H-3’), 4.12 (t, 2H, J = 8.8 Hz, H-2, H-5), 4.28 (s, 1H, H-4), 4.42 (d, 1H, J = 11.2 Hz, CHHPh), 4.50 (d, 1H, J = 11.2 Hz, CHHPh), 4.63 (d, 1H, J = 11.8 Hz, CHHPh), 4.64 (d, 1H, J = 11.4 Hz, CHHPh), 4.72 (d, 1H, J = 11.2 Hz, CHHPh), 4.80 (d, 1H, J = 7.6 Hz, H-1), 4.84 (d, 1H, J = 11.2 Hz, CHHPh), 4.90 (d, 1H, J = 1.2 Hz, H-5’), 5.04 (d, 1H, J = 11.6 Hz, CHHPh), 5.08 (d, 1H, J = 11.2 Hz, CHHPh), 5.27 (d, 1H, J = 3.2 Hz, H-1’), 5.50 (bs, 1H, H-4’), 6.77 – 7.43 (m, 24H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.1 (CH3 COOMe), 52.4 (CH3 COOMe), 55.6 (CH3 pMP), 68.6 (C-4’), 69.0 (C-5’), 71.9 (CH2 Bn), 73.9 (C-4), 74.0 (C-5), 74.4 (C-3 or C-2’), 74.7 (CH2 Bn), 74.9 (CH2 Bn), 75.4 (CH2 Bn), 75.6 (C-3’), 76.4 (C-2’ or C-3), 76.9 (C-2), 95.3 (C-1’), 103.3 (C-1), 114.4 (CH Arom pMP), 118.8 (CH Arom pMP), 127.2 – 128.5 (CH Arom), 137.7 (Cq Bn), 137.9 (Cq Bn), 138.3 (Cq Bn), 151.3 (Cq pMP), 155.4 (Cq pMP), 168.0 (C=O Ac or COOMe), 168.1 (C=O Ac or COOMe), 169.7 (C=O Ac or COOMe); HRMS: [M+H]+ calcd for C51H55O15 924.38010, found 924.38050.

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45

Chapter 2    Methyl (phenyl 4-O-acetyl-2,3-di-O-benzoyl-1-thio-β-D-glucopyranoside) uronate (57): A solution of 138 mg compound 28 (0.3 mmol) in 2 mL pyridine and 0.7 mL Ac2O was OBz stirred for 3h before 3 mL MeOH was added. The reaction mixture was concentrated under reduced pressure and several times co-evaporated with toluene. Flash column chromatography using AcO BzO

COOMe O

SPh

EtOAc/petroleum ether afforded 153 mg of the title compound 57 (0.3 mmol, quant.) as a colorless oil. TLC: 25% EtOAc/PE; MP: 178°C; [α]D22: +59° (c = 1, CHCl3); IR (neat, cm-1): 1066, 1093, 1226, 1257, 1708; 1H NMR (400 MHz, CDCl3) δ = 1.94 (s, 3H, CH3 Ac), 3.80 (s, 3H, CH3 COOMe), 4.23 (d, 1H, J = 10.0 Hz, H-5), 5.99 (d, 1H, J = 10.0 Hz, H-1), 5.40 (t, 1H, J = 10.0 Hz, H-2), 5.45 (t, 1H, J = 10.2 Hz, H-4), 5.73 (t, 1H, J = 9.4 Hz, H-3), 7.31 – 7.96 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.4 (CH3 Ac), 52.9 (CH3 COOMe), 69.4 (C-4), 69.9 (C-2), 73.6 (C-3), 76.4 (C-5), 86.5 (C-1), 128.4 – 133.4 (CH Arom), 128.5 (Cq SPh), 131.3 (Cq Bz), 164.8 (C=O Ac, Bz or COOMe), 165.5 (C=O Ac, Bz or COOMe), 166.9 (C=O Ac, Bz or COOMe); HRMS: [M+H]+ calcd for C29H27O9S 551.13703, found 551.13885.

AcO BzO

COOMe O

Methyl 2,3,4-tri-O-benzyl-6-O-(methyl 4-O-acetyl-2,3-di-O-benzoyl-β-D-glucoO

OMe OBn

pyranosyluronate)-α-D-glucopyranoside (58): Donor 57 was glycosylated with O OBz acceptor 4817 in the same way as described in the general procedure using OBn BnO BSP/Tf2O delivering 57 mg of the title compound 58 (0.063 mmol, 63%). NB: In this case TTBP was excluded from the reaction mixture to prevent ortho-ester formation. TLC: 20% EtOAc/toluene; MP: 168°C (decomp.); [α]D22: +40° (c = 1, CHCl3); IR (neat, cm-1): 1024, 1066, 1091, 1220, 1276, 1730; 1H NMR (400 MHz, CDCl3) δ = 1.94 (s, 3H, CH3 Ac), 3.18 (s, 3H, CH3 OMe), 3.35 (m, 1H, J = 9.2 Hz, H-4), 3.40 (dd, 1H, J = 9.6 Hz, J = 3.2 Hz, H-2), 3.69 (m, 2H, H-5, H-6), 3.76 (s, 3H, CH3 COOMe), 4.00 (t, J = 9.6 Hz, 1H, H-3), 4.14 (m, 2H, H-5’, H-6), 4.31 (d, 1H, J = 11.2 Hz, CHHPh), 4.47 (d, 1H, J = 3.6 Hz, H-1), 4.56 (d, 1H, J = 8.4 Hz, CHHPh), 4.58 (d, 1H, J = 9.6 Hz, CHHPh), 4.68 (d, 2H, J = 10.8 Hz, 2xCHHPh), 4.74 (d, 1H, J = 6.8 Hz, H-1’), 4.90 (d, 1H, J = 10.8 Hz, CHHPh), 5.45 – 5.51 (m, 2H, J = 9.6 Hz, J = 6.4 Hz, H-2’, H-4’), 5.68 (t, 1H, J = 9.4 Hz, H-3’), 7.08 – 7.91 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.4 (CH3 Ac), 52.8 (CH3 COOMe), 54.9 (CH3 OMe), 68.5 (C-6), 69.3 (C-2’ or C-4’), 69.5 (C-5), 71.4 (C-4’ or C-2’), 72.2 (C-3’), 72.7 (C-5’), 73.2 (CH2 Bn), 74.6 (CH2 Bn), 75.4 (CH2 Bn), 77.4 (C-4), 79.7 (C-2), 81.8 (C-3), 97.8 (C-1), 101.1 (C-1’), 127.4 – 133.4 (CH Arom), 128.3 (Cq Bz), 129.0 (Cq Bz), 138.1 (Cq Bn), 138.7 (Cq Bn), 163.2 (C=O Bz or Ac or COOMe), 164.7 (C=O Bz or Ac or COOMe), 165.6 (C=O Bz or Ac or COOMe), 167.1 (C=O Bz or Ac or COOMe), 169.2 (C=O Bz or Ac or COOMe); HRMS: [M+Na]+ calcd for C51H52O15Na 927.31984. found 927.32434.

OAc

Methyl (ethyl 2,3-di-O-benzyl-4-O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-

α-D-glucopyranosyl)-1-thio-β-D-glucopyranoside) uronate (60): A solution of 128 COOMe mg donor 59 (0.3 mmol), 170 mg diphenylsulfoxide (0.84 mmol, 2.5 equiv.) and N3 O O SEt BnO 223 mg tri-tert-butylpyrimidine (0.9 mmol, 3 equiv.) in 3 mL DCM was stirred OBn over 100 mg activated MS3Å for 30 min. The mixture was cooled to -60°C before 69 µL triflic acid anhydride (0.42 mmol, 1.4 equiv.) was added. The mixture was allowed to warm to -40°C in BnO BnO

O

1h followed by addition of 173 mg acceptor 13 (0.4 mmol, 1.5 equiv.) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to 0°C. Subsequently 0.16 mL triethylamine was added to the reaction mixture. Flash chromatography (10% EtOAc/PE) and removal of the eluent afforded 169 mg of the title compound 60 (0.2 mmol, 67%) as a colorless oil. TLC: 20% EtOAc/toluene; [α]D22: +15° (c = 1, CHCl3); IR (neat, cm-1): 1028, 1074, 1209, 1236, 1361, 1444, 1741, 2108; 1H NMR (400 MHz, CDCl3) δ = 1.23 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.01 (s, 3H, CH3 Ac), 2.73 – 2.78 (m, 2H, CH2 SEt), 3.50 (t, 1H, J = 9.2 Hz, H-2), 3.52 (t, 1H, J = 8.8 Hz, H-4’), 3.58 (bd, 1H, J = 10.0 Hz, H-5’), 3.75 (s, 3H, CH3 COOMe), 3.78 (t, 1H, J = 8.8 Hz, H-3),

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Thioglycuronides: Synthesis and Application    3.88 (t, 1H, J = 9.2 Hz, H-3’), 3.91 (d, 1H, J = 9.6 Hz, H-5), 4.13 (t, 1H, J = 9.4 Hz, H-4), 4.28 (bs, 2H, H-6’), 4.51 (t, 2H, J = 9.4 Hz, H-1, CHHPh), 4.65 (d, 1H, J = 10.0 Hz, CHHPh), 4.80 (d, 1H, J = 11.2 Hz, CHHPh), 4.86 (s, 2H, CH2 Bn), 4.90 (d, 1H, J = 10.8 Hz, CHHPh), 4.93 (d, 1H, J = 10.4 Hz, CHHPh), 5.03 (d, 1H, J = 10.8 Hz, CHHPh), 5.53 (d, 1H, J = 3.6 Hz, H-1’), 7.24 – 7.71 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 14.8 (CH3 SEt), 20.8 (CH3 Ac), 25.0 (CH2 SEt), 52.7 (CH3 COOMe), 62.1 (C-6’), 63.2 (C-2’), 69.6 (C-5’), 74.9 (CH2 Bn), 75.1 (C-4), 75.1 (CH2 Bn), 75.2 (CH2 Bn), 75.4 (CH2 Bn), 77.4 (C-4’), 77.8 (C-5), 80.0 (C-3’), 81.2 (C-2), 85.5 (C-3), 85.6 (C-1), 97.7 (C-1’); 124.7 – 131.0 (CH Arom), 137.5 (Cq Bn), 138.1 (Cq Bn), 168.3 (C=O Ac or COOMe), 170.6 (C=O Ac or COOMe), ESI-MS: 864.4 [M+Na]+. 3-O-Benzoyl-5,6-O-isopropylidene-D-galactono-1,4-lactone (61): Commercially available (910 mg, 5 mmol) was suspended in 25 mL acetone before 572 mg O dimethoxypropane (5.5 mmol, 1.1 equiv.) and a catalytic amount of pTsOH were added. BzO OH The mixture was stirred overnight, neutralized with 5 mL NEt3 and concentrated under reduced pressure. The crude oil was dissolved in 25 mL DCM and cooled to 0°C. Subsequently 408 mg O

H

O

O

D-galactono-1,4-lactone

imidazole (6 mmol, 1.2 equiv.) and 830 mg TBDMSCl (5.5 mmol, 1.1 equiv.) were added. The mixture was stirred for 15 min and filtered over a plug of silica gel. The filtrate was concentrated under reduced pressure. The crude oil was dissolved in 25 mL pyridine and cooled to 0°C before 0.58 mL BzCl (5 mmol, 1 equiv.) was added. The reaction mixture was allowed to stir overnight followed by the addition of 1 mL MeOH. The mixture was concentrated and filtered over a short plug of silica gel. Evaporation of the solvent yielded the completely protected intermediate. This was dissolved in 20 mL THF and treated with 0.29 mL AcOH and 5 mL TBAF (5 mmol, 1 equiv.) for 6 h. The reaction mixture was diluted with EtOAc and washed with NaHCO3 (sat. aq.) and brine. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography, using EtOAc/petroleum ether afforded 656 mg of the title compound 61 (2.10 mmol, 62% over the 4 steps) as a colorless oil. TLC: 40% EtOAc/toluene; [α]D22: +24° (c = 1, CHCl3); IR (neat, cm-1): 636, 694, 756, 889, 912, 1008, 1029, 1157, 1271, 1444, 1488, 1728, 1793; 1H NMR (400 MHz, CDCl3) δ = 1.58 (s, 3H, CH3 isoprop), 1.60 (s, 3H, CH3 isoprop), 3.94 (d, 1H, J = 8.4 Hz, OH-2), 4.05 (t, 1H, J = 6.8 Hz, H-6), 4.19 (dd, 1H, J = 8.4 Hz, J = 6.7 Hz, H-6), 4.51 – 4.57 (m, 3H, H-2, H-4, H-5), 5.42 (t, 1H, J = 3.2 Hz, H-3), 7.42 – 7.86 (m, 5H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 25.5 (CH3 isoprop), 25.6 (CH3 isoprop), 65.2 (C-6), 71.9 (C2), 74.7 (C-5), 77.9 (C-3), 79.7 (C-4), 111.1 (Cq isoprop), 128.7 – 134.1 (CH Arom), 172.3 (C=O lactone); ESIMS: 323.0 [M+H]+; HRMS: [M+H]+ calcd for C16H23O7N 340.1391, found 340.1380. OAc BnO BnO

O N3 O BnO

COOMe O BnO

OBz O

O

O

O

H

O

3-O-Benzoyl-2-O-(methyl 4-O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranosyl)-2,3-di-Obenzyl-α-D-glucopyranosyluronate)-5,6-O-isopropylidene-D-galactono-1,4-lactone (62): Donor 60 was glycosylated with acceptor 61 in the same way as described in the general procedure using BSP/Tf2O delivering 38 mg of the title compound 62 (0.035 mmol, 35%) as a colorless oil (α/β = 4/1). TLC: 20% EtOAc/toluene; IR (neat, cm-1): 1174, 1296, 1676, 1743, 2106; α-anomer: 1H NMR (600 MHz, CDCl3) δ = 1.41 (s, 3H, CH3 isoprop), 1.43 (s, 3H, CH3 isoprop), 2.00 (s, 3H, CH3 Ac), 3.19 (dd, 1H, J = 10.4 Hz, J = 3.7 Hz, H-2”), 3.44 – 3.45 (m, 2H, H-4”, H-5”), 3.45 (s, 3H, CH3 COOMe), 3.66 (t, 1H, J = 8.2 Hz, H-2’), 3.68 – 3.71 (m, 2H, J = 9.5 Hz, J = 9.4 Hz, H-2’, H-3”), 3.97 (dd, 1H, J = 8.5 Hz, J = 6.5 Hz, H-6), 4.00 (t, 1H, J = 9.3 Hz, H-4’), 4.08 (t,

47   

47

Chapter 2    1H, J = 9.1 Hz, H-3’), 4.11 – 4.14 (m, 2H, H-6, H-6”), 4.20 – 4.23 (m, 2H, H-5’, H-6”), 4.43 (dd, 1H, J = 5.9 Hz, J = 2.9 Hz, H-4), 4.51 (d, 1H, J = 10.9 Hz, CHHPh), 4.56 (dt, 1H, J = 6.7 Hz, J = 2.8 Hz, H-5), 4.65 (d, 1H, J = 11.3 Hz, CHHPh), 4.71 (d, 1H, J = 10.5 Hz, CHHPh), 4.73 (d, 1H, J = 10.5 Hz, CHHPh), 4.79 (d, 1H, J = 10.9 Hz, CHHPh), 4.81 (d, 1H, J = 6.7 Hz, H-2), 4.82 (d, 1H, J = 10.3 Hz, CHHPh), 4.84 (d, 1H, J = 11.0 Hz, CHHPh), 5.08 (d, 1H, J = 10.6 Hz, CHHPh), 5.52 (d, 1H, J = 3.7 Hz, H-1”), 5.61 (d, 1H, J = 3.5 Hz, H-1’), 5.89 (t, 1H, J = 6.5 Hz, H-3”), 7.23 – 8.09 (m, 25H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 20.8 (CH3 Ac), 25.4 (CH3 isoprop), 25.9 (CH3 isoprop), 52.4 (CH3 COOMe), 62.2 (C-6”), 63.1 (C-2”), 65.1 (C-6), 69.5 (C-5’’), 70.5 (C-5’), 72.4 (CH2 Bn), 74.3 (C-3), 74.6 (C-2, C-5), 74.8 (CH2 Bn), 74.9 (C-4’), 75.2 (CH2 Bn), 75.4 (CH2 Bn), 77.4 (C-4”), 78.6 (C-2’), 80.0 (C-4), 80.7 (C-3”), 81.0 (C-3’), 96.1 (C-1’), 97.9 (C-1”), 110.7 (Cq isoprop), 125.2 – 134.0 (CH Arom), 137.2 (Cq Bn), 137.6 (Cq Bn), 138.1 (Cq Bn), 138.4 (Cq Bn), 165.4 (C=O Ac, Bz or COOMe), 169.1 (C=O Ac, Bz or COOMe), 170.7 (C=O Ac, Bz or COOMe); HRMS: [M+H]+ calcd for C59H64O18N3 1119.44449, found 1119.44702.

References and Notes 1

Published in part: Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168.

2

Glycochemistry: Principles, Synthesis, and Applications, Wang, P.G.; Bertozzi, C.P. Eds.; Marcel Dekker: New York, 2001; pp 425–492. (a) For a review on the use of thioglycosides as glycosyl donors: Garegg, P.J. Adv. Carbohydr. Chem. Biochem. 1997, 52, 179–205 (b) Davis, B.G. J. Chem. Soc. Perkin Trans. 1 2000, 14, 2137–2160.

3 4 5 6 7 8 9

See Chapter 1 and references cited therein. Codée, J.D.C; Van der Marel, G.A.; Van Boeckel, C.A.A.; Van Boom, J.H. Eur. J. Org. Chem. 2002, 23, 3954–3965. Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950. (a) Garcia, B.A.; Poole, J.L.; Gin, D.Y.; J. Am. Chem. Soc. 1997, 119, 7597–7598 (b) Garcia, B.A.; Gin, D.Y.; J. Am. Chem. Soc. 2000, 122, 4269–4279. Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A.; Org. Lett. 2003, 5, 1519–1522.

10 De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G. J. Org. Chem. 1997, 62, 6974–6977. 11 High concentrations of water and two equivalents of BAIB facilitate the conversion of the aldehyde into the respective carboxylic acid (Epp, J.B.; Widlanski, T.S. J. Org. Chem. 1999, 64, 293–295). See also ref 10. 12 Garegg, P.J.; Kvarnstroem, I.; Niklasson, A.; Niklasson, G.; Svensson, S.C.T. J. Carbohydr. Chem. 1993, 12, 933–954. 13 Suzuki, K.; Ohtsuka, I.; Kanemitsu, T.; Ako, T.; Kanie, O. J. Carbohydr. Chem. 2005, 24, 219–236. 14 Lefeber, D.J.; Arevalo, E.A.; Kamerling, J.P.; Vliegenthart, J.F.G. Can. J. Chem. 2002, 80, 76–81. 15 Ziegler, T.; Eckhardt, E.; Herold, G. Liebigs Ann. Chem. 1992, 441–452. 16 Activation of the donor for 5 min. at –60°C revealed incomplete activation of the donor glycoside. Activation was started at –60°C and the reaction mixture was gradually warmed to –40°C within 15 min (see also ref. 8). 17 Xu, L.; Price, N.P.J. Carbohydr. Res. 2004, 339, 1173–1178. 18 Crich, D.; Bowers, A.A. J. Org. Chem. 2006, 71, 3452–3463.

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Thioglycuronides: Synthesis and Application    19 Krog-Jensen, C.; Oscarson, S. Carbohydr. Res. 1998, 308, 287–296. 20 (a) Iwahara, S.; Suemori, N.; RamLi, N.; Takegawa, K. Biosci. Biotech. Biochem. 1995, 59, 1082–1085 (b) Jikibara, T.; Takegawa, K.; Iwahara, S. J. Biochem. 1992, 111, 225–229. 21 Codée, J.D.C.; Stubba, B.; Schiattarella, M.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. J. Am. Chem. Soc. 2005, 127, 3767–3773. 22 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis, 2001, 323–326. 23 See experimental part for method of synthesis. 24 Fales, H.M.; Jaouni, T.M.; Babashak, J.F. Anal. Chem. 1973, 45, 2302–2303. 25 Magaud, D.; Grandjean, C.; Doutheau, A.; Anker, D.; Shevchik, V.; Cotte-Pattat, N.; Robert-Baudouy, J. Carbohydr. Res.; 2000, 314, 189–199.

49   

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50

 

   

Chapter 3 │  Preparation of 1‐Thio Uronic     Acid Lactones and Their Use in    Oligosaccharide Synthesis

Abstract: The chemo- and regioselective TEMPO/BAIB-mediated oxidation of 2,6- and 3,6dihydroxy 1-thio glycopyranosides to the corresponding 1-thio uronic acid lactones is described. These locked 1-thio glycuronides can directly be used as donors in glycosidation reactions using the Ph2SO/Tf2O reagent system. Alternatively, selective opening of the lactone bridge liberates a hydroxyl function for ensuing glycosylations.1

Introduction The 2,2,6,6-tetramethylpiperidinyloxy free radical 1 (TEMPO, Figure 1A) is a versatile and often applied reagent for the selective oxidation of primary hydroxyl functions.2 The active species in this reaction is believed to be the N-oxoammonium salt 2, generated in situ from the reaction between TEMPO (1) and any of a number of co-oxidants, including mchloroperoxybenzoic acid,3 calcium or sodium hypochlorite4 and several hypervalent iodine(III) species.5 Recently, it was reported that the combination of TEMPO and [bis(acetoxy)iodo]benzene (BAIB) enabled the chemo- and regioselective oxidation of primary alcohols into the corresponding aldehydes6 or carboxylic acids.7 The Forsyth group used the same reagent combination for the synthesis of δ-lactones from the corresponding 1,5diol systems having a primary and a secondary hydroxyl function.8 Studies on this process revealed the formation of a lactol intermediate via selective oxidation of the primary hydroxyl function followed by nucleophilic attack of the secondary alcohol on the resulting aldehyde. In the next oxidation cycle, the lactol is oxidized to the lactone.9 51   

51

Chapter 3   

Chapter 2 describes the application of the TEMPO/BAIB reagent system in the chemo- and regioselective oxidation of a variety of 4,6-dihydroxy 1-thioglycosides (3, Figure 1B).10,11 The 1-thio uronic esters 5, obtained by treatment of 4 with diazomethane, proved to be valuable building blocks in oligosaccharide synthesis. In an extension of this method for the selective oxidation of 4,6-dihydroxy 1-thioglycosides, this Chapter outlines the tandem oxidationlactonization process of a variety of 2,6- and 3,6-dihydroxy 1-thioglycosides and application of the resulting 6,2- and 6,3-lactones in oligosaccharide synthesis.

Figure 1. A

O

O

N

OH O

2

(OP)n

DCM H 2O

HO

3

I

SR

O

O Ac

BAIB

2

TE MPO (1) B

O Ac

N

BA IB

OH

CH 2N2

O

HO

(OP)n 4

DMF SR

O HO

OMe O (O P)n

SR

5

Results and Discussion In a first attempt to induce lactone formation, phenyl 2,4-di-O-benzyl-1-thio-β-Dgalactopyranoside (6)12 was treated with 0.2 equiv. TEMPO and 2.5 equiv. BAIB in anhydrous DCM (Table 1, entry 1). The expected 6,3-lactone13 7 was obtained in 54% yield along with a considerable amount of sulfoxide and sulfone by-products. Changing the reaction medium to the biphasic DCM/H2O system led to the rapid and efficient transformation of diol 6 into lactone 7 (75% yield) with only trace amounts of the side products formed. Subjection of ethyl 3,4-di-O-benzyl-1-thio-α-D-mannopyranoside (9), which has a 2,6-cis diol configuration, to the latter oxidation conditions afforded lactone product 10 in 77% yield (entry 2). Similarly, glycopyranosides 12 and 15 were converted into lactones 13 and 16 (entries 3 and 4). The relatively poor yield of lactone 16 is likely due to the change from the “all equatorial” 4C1 conformation of compound 15 into its 1C4 conformation in which all substituents are axially oriented. As a final example, compound 18 containing an anomeric hydroxyl group was converted into the corresponding 6,1-lactone 19, albeit in moderate yield (27%). The next objective was to find suitable conditions for selective opening of the lactone bridge, liberating the hydroxyl function for potential functionalization in ensuing glycosylation events. Stirring compounds 10 and 13 in anhydrous methanol under gentle

52   

52

1‐Thio Uronic Acid Lactones in Oligosaccharide Synthesis   

reflux furnished the corresponding transesterified products 11 and 14 (entries 2 and 3) in a quantitative yield. As exemplified in entry 3, the relatively labile acetyl group is stable with respect to the cleavage conditions of the lactone bridge.14 The lactone function in compounds 7 and 16 (entry 1 and 4) proved to be stable to these conditions.15 However, stirring 7 and 16 in methanol containing a catalytic amount of p-toluenesulfonic acid delivered the desired esters 8 and 17 in a quantitative yield. The conformationally locked lactones 7, 10, 13, and 16 have potential for stereoselective glycosylation reactions.16 It is now well established that the conformation of donor glycosides plays a pivotal role in governing both reactivity and anomeric selectivity in glycosidations.17 Table 1.

entry

substrate

yielda

lactone

yielda

ester

O

BnO

1

HO

OH

SPh

O

O

SPh

54%b

O

BnO

c

OBn 6

OBn

75%

BnO

COOMe O

HO

SPh

quant.e

OBn 8

7

O

2

HO BnO BnO

OH O

O

O

BnO BnO SEt

77%c

MeOOC OH O BnO BnO SEt

SEt 10

9

quant.d

11

O

3

HO BnO HO

O

OAc O SEt

12

SEt OAc

O

72%c

MeOOC BnO HO

OAc O

BnO

quant.d

SEt

13

14

O OH

4

O

BnO HO

SPh

O SPh

OBn

BnO

15

51%c

O

BnO HO

COOMe O

SPh

quant.e

OBn

OBn

17

16

O

OH

5

BnO BnO

BnO 18 a

OBn O

O OH

BnO

O

27%c

---

---

OBn 19

isolated yields. b 0.2 equiv. TEMPO, 2.5 equiv. BAIB, DCM. DCM/H2O (2/1). d MeOH, reflux. d TsOH, MeOH, rt.

c

0.2 equiv. TEMPO, 2.5 equiv. BAIB,

53   

53

Chapter 3   

For example, Fraser-Reid and coworkers reported that benzylidene protected glycosides are less reactive than their benzylated counterparts.18 This difference in reactivity was attributed to so-called “torsional disarmament” and has shown promise in increasing anomeric selectivities.19 On the other hand, it has been stated that in glycosylation events torsional effects are, in general, overruled by the electronic effects exerted by the ring substituents.20 However, Bols and coworkers demonstrated that in the case of 1C4 locked methyl 3,6anhydro-β-D-glycosides, the reactivity of the anomeric function is influenced by electronic effects originating from the orientation of the remaining hydroxyl substituents.21 Chapter 2 describes the decreased nucleophilicity of the sulfur atom at the anomeric centre due to the remotely attached carboxylic ester at the C6 position.10 The intrinsic low reactivity of the Table 2.

entry

donor

activator yield (α/β)a,d

acceptor

disaccharide O

O SPh

O

1

O

BnO

OH

Ph2SO/Tf2Ob 69% (1/0)

O

BnO BnO

BnO

OBn 7

20

O

O O

BnO

OMe OBn

O BnO OBn OBn 21

OMe

O

OH

2

7

Ph2SO/Tf2Ob 98% (1/0)

O

AcO AcO

AcO 22

O

O O

BnO

OMe OAc

O AcO OAc OBn 23

OMe

O

3

7

COOMe O

HO BnO

BnO 24

Ph2SO/Tf2Ob 91% (1/0)

O O

BnO

O BnO

COOMe O BnO

OBn

OMe

OMe

25 O

OBn

4

7

Ph2SO/Tf2Oc 69% (1/0)

O

HO BnO

CbzHN 26

OBn

O

CbzHN

OBn 27

OMe

O

O BnO

O

BnO

OMe

OpMP

HO

5

7

COOMe O

BnO OBn

OpMP

Ph2SO/Tf2Ob 50% (4/1)

28 a

isolated yields. spectroscopy.

54   

54

b

2.5 equiv. TTBP.

c

O

O

1 equiv. TTBP.

MeOOC O

O BnO

O

OBn

BnO OBn 29

d

anomeric ratios were determined by 1H NMR

1‐Thio Uronic Acid Lactones in Oligosaccharide Synthesis   

1-thio methyl uronates was overcome by activation with the highly electrophilic Ph2SO/Tf2O reagent combination at slightly elevated temperature (-40°C) compared to the standard activation temperature (-60°C).22 Galactosyl donor 7 was chosen as a model compound to determine the influence of the bridged lactone function in glycosidation reactions (Table 2). Treatment of lactone 7 with 1.3 equiv. Ph2SO, 1.3 equiv. Tf2O and 2.5 equiv. TTBP23 at -50°C gave full activation within 15 min. Addition of acceptor 20,24 bearing a primary hydroxyl group afforded disaccharide 21 in good yield (entry 1). The heteronuclear coupling constant between C1 and H1 (so-called 13C-GATED experiment) revealed the presence of an α-interglycosidic linkage. Treatment of an activated mixture of donor 7 with acceptor 22 afforded disaccharide 23 in near quantitative yield, again fully α-selective (entry 2).25 Analogously, methyl uronate 24 was applied as acceptor species in the Ph2SO/Tf2O-mediated glycosidation involving donor 7, resulting in α-selective formation of disaccharide 25 (entry 3). Changing the acceptor towards N-(benzyloxycarbonyl)-protected glucosamine 26 also delivered the corresponding disaccharide 27 as a single diastereomer (entry 4). This reaction proceeded slower than the other glycosidations, probably due to reduced reactivity of the acceptor alcohol acting as an intramolecular H-bond acceptor.26 The amount of base (tri-tertbutylpyrimidine, TTBP)27 used in this condensation was reduced to one equivalent with respect to the amount of Tf2O to circumvent possible side-reactions involving the nucleophilic N-atom.28 Performing the reaction in the absence of base gave no significant improvement in yield. Upon addition of galactopyranose 28 to the activated mixture of donor 7, Ph2SO and Tf2O, a 4/1 mixture of α- and β-disaccharide 29 was formed (entry 5). The reduced stereoselectivity may be related to the steric environment of the OH4 group in acceptor 28, hampering the formation of the α-product.29

Scheme 1. O

BnO

BnO

OBn

O

CbzHN

OBn 27

AcO TBSO

O

O BnO

O

a

HO

COOMe O BnO

OMe

OBn O

O BnO

30

CbzHN

OBz O

BnO O

AcO COOMe O

OBz

BnO 32

O BnO

TBSO OBn

31

OMe

OBz O

SPh b

OBz

O CbzHN

OMe

Reagents and conditions: (a) KOtBu, MeOH, 82% (b) 31, Ph2SO, Tf2O, DCM, -60°C then 30, 63%.

55   

55

Chapter 3   

Having established that locked 1-thio uronic acid lactones can be used as donors in glycosylation reactions it was decided to explore their acceptor properties after the opening of the lactone bridge. Disaccharide 27 was taken as a model compound towards the synthesis of nonnatural trisaccharide 32 (Scheme 1). Treatment of benzylated lactone 27 with potassium tert-butoxide in MeOH under strictly anhydrous conditions afforded the desired acceptor 30 in a yield of 82%. Pre-activation of 2-O-acylated donor 31 using diphenylsulfonium bistriflate at -60°C in DCM followed by addition of acceptor 30 afforded trisaccharide 32 in 63% yield. The regular acid scavenger TTBP was excluded from the reaction mixture to prevent putative ortho-ester formation.

Conclusion This Chapter describes an efficient synthetic route for locked 1-thio 6,2- and 6,3-lactones and their implementation as both donor and acceptor in Ph2SO/Tf2O-mediated coupling reactions. Compared to the open-form galacturonic acid esters decribed in Chapter 2, the galacto-6,3-lactones possess higher reactivity and superior stereoselectivity in glycosylation. Application of these lactones in oligosaccharide synthesis is shown for the synthesis of trisaccharide 32 requiring only a minimal number of protective group manipulations at the disaccharide stage.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Brüker DPX-300 (300/75 MHz), Brüker AV400 (400/100 MHz) and a Brüker DMX-600 (600/150 MHz) spectrometer. Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and Q-Star Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide and TTBP were removed by co-evaporation with toluene. TTBP27 was synthesised as described by Crich et al. Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled immediately prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography and TLC were of technical grade and distilled before use. Flash chromatography was performed on Fluka silica gel 60 (0.04 – 0.063 mm). TLCanalysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in ethanol followed by charring at ~150°C.

56   

56

1‐Thio Uronic Acid Lactones in Oligosaccharide Synthesis    General Procedure for the TEMPO/BAIB-Mediated Tandem Oxidation/Lactonization: To a vigorously stirred solution of 0.3 mmol thioglycoside in 1 mL DCM and 0.5 mL H2O was added 0.06 mmol TEMPO (0.2 equiv.) and 0.75 mmol BAIB (2.5 equiv.). Stirring was allowed until TLC indicated complete conversion of the starting material to a higher running spot (~15min). The reaction mixture was quenched by the addition of 10 mL Na2S2O3 solution (10% in H2O) and 10 mL NaHCO3 (sat., aq.). The mixture was then extracted twice with EtOAc (10 mL) and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash column chromatography using EtOAc/petroleum ether afforded the pure lactones. General Procedure for Glycosidations using Ph2SO/Tf2O: A solution of lactone (1 equiv), diphenyl sulfoxide (1.3 equiv.) and tri-tert-butylpyrimidine (2.5 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30 min. The mixture was cooled to -60°C before triflic acid anhydride (1.3 equiv.) was added. The mixture was allowed to warm to -50°C in 15min followed by addition of acceptor (1.5 equiv.) in DCM (0.15M). Stirring was continued and the reaction mixture was allowed to warm to 0°C. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding coupled products. Phenyl 2,4-di-O-benzyl-1-thio-β-D-galactopyranosidurono-6,3-lactone (7): Diol 6 was conver-

O SPh

ted according to the general procedure delivering lactone 7 in 75% yield as a colorless oil. O TLC: 20% EtOAc/PE; [α]D22: -95° (c = 0.5, CHCl3); IR (neat, cm-1): 979, 1026, 1060, 1097, BnO 1151, 1365, 1454, 1799, 2869, 3030; 1H NMR (400 MHz, CDCl3) δ = 4.02 (s, 1H, H-4), 4.23 OBn (d, 1H, J = 4.8 Hz, H-2), 4.36 (s, 1H, H-5), 4.48 (d, 1H, J = 11.8 Hz, CHHPh), 4.51 (s, 2H, CH2Ph), 4.56 (d, 1H, J = 11.8 Hz, CHHPh), 4.79 (d, 1H, J = 4.4 Hz, H-3), 5.40 (s, 1H, H-1), 7.14 – 7.43 (m, 15H, H Arom); 13C NMR O

(100 MHz, CDCl3) δ = 70.6 (C-4), 71.1 (CH2 Bn), 72.7 (CH2 Bn), 75.7 (C-5), 78.4 (C-2), 78.6 (C-3), 85.6 (C-1), 127.6 – 132.4 (CH Arom), 133.5 (Cq SPh), 136.4 (Cq Bn), 136.5 (Cq Bn), 172.5 (C=O); 13C-GATED (125 MHz, CDCl3): 85.6 (J = 168 Hz, C-1); HRMS: [M+NH4]+ calcd for C26H28O5SN 466.16882, found 466.16946. BnO

Methyl (phenyl 2,4-di-O-benzyl-1-thio-β-D-galactopyranoside) uronate (8): Compound 7 was

COOMe O

suspended in MeOH and a catalytic amount TsOH was added. The mixture was stirred for 3h HO SPh OBn until TLC-analysis indicated complete conversion. The reaction mixture was neutralized by adding 0.1 mL Et3N, followed by concentration under reduced pressure. Flash chromatography and removal of the eluent afforded the title compound 8 (quant.) as a colorless oil. TLC: 20% EtOAc/PE; IR (neat, cm-1): 1026, 1103, 1348, 1438, 1735, 2858; 1H NMR (400 MHz, CDCl3) δ = 2.26 (d, 1H, J = 4.8 Hz, OH-3), 3.70 (t, 1H, J = 8.8 Hz, H-2), 3.73 (m, 4H, H-3 and CH3 COOMe), 4.10 (s, 1H, H-5), 4.23 (d, 1H, J = 1.2 Hz, H-4), 4.57 (d, 1H, J = 8.8 Hz, H-1), 4.63 (d, 2H, J = 11.2 Hz, 2xCHHPh), 4.70 (d, 1H, J = 11.2 Hz, CHHPh), 4.91 (d, 1H, J = 11.2 Hz, CHHPh), 7.24 – 7.66 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.4 (CH3 COOMe), 74.8 (CH2 Bn), 75.3 (CH2 Bn), 75.3 (C-3), 77.0 (C-5), 77.3 (C-4), 77.5 (C-2), 87.5 (C-1), 127.6 – 132.3 (CH Arom), 133.5 (Cq SPh), 137.9 (Cq Bn), 138.0 (Cq Bn), 168.4 (C=O); HRMS: [M+Na]+ calcd for C27H28O6SNa 503.14988, found 503.15082. O O BnO BnO

O

Ethyl 3,4-di-O-benzyl-1-thio-α-D-mannopyranosidurono-6,2-lactone (10): Diol 9 was converted according to the general procedure delivering lactone 10 in 77% yield as a colorless oil. TLC:

20% EtOAc/PE; [α]D22: +152° (c = 1.7, CHCl3); IR (neat, cm-1): 981, 1064, 1188, 1352, 1454, SEt 1672, 1778, 2871, 3031, 3192; 1H NMR (300 MHz, CDCl3) δ = 1.34 (t, 3H, J = 7.4 Hz, CH3 SEt), 2.77 (m, 2H, CH2 SEt), 3.80 (s, 1H, H-4), 4.07 (s, 1H, H-3), 4.47 (d, 1H, J = 11.2 Hz, CHHPh), 4.49 (s,

57   

57

Chapter 3    1H, H-5), 4.50 (d, 1H, J = 12.4 Hz, CHHPh), 4.61 (d, 1H, J = 11.2 Hz, CHHPh), 4.65 (d, 1H, J = 12.4 Hz, CHHPh), 4.85 (s, 1H, H-2), 5.27 (d, 1H, J = 2.6 Hz, H-1), 7.30 – 7.36 (m, 10H, H Arom); 13C NMR (75 MHz, CDCl3) δ = 16.1 (CH3 SEt), 25.8 (CH2 SEt), 71.8 (CH2 Bn), 71.9 (CH2 Bn), 72.9, 76.3, 77.9, 80.6 (C-2, C-3, C4, C-5), 81.6 (C-1), 128.7 – 129.4 (CH Arom), 137.6 (Cq Bn), 137.7 (Cq Bn), 168.8 (C=O); 13C-GATED (75 MHz, CDCl3): 81.6 (J = 169 Hz, C-1); HRMS: [M+NH4]+ calcd for C22H28O5SN 418.16827, found 418.16864. MeOOC BnO BnO

OH O SEt

Methyl (ethyl 3,4-di-O-benzyl-1-thio-α-D-mannopyranoside) uronate (11): Compound 10 was suspended in MeOH and stirred at reflux temperature for 5h after which TLC-analysis indicated complete conversion. Removal of the methanol under reduced pressure and co-

evaporation of the residual oil using chloroform afforded the title compound 11 (quant.) as a light yellow oil. TLC: 20% EtOAc/PE; [α]D22: +72° (c = 1, CHCl3); IR (neat, cm-1): 702, 732, 1033, 1087, 1234, 1373, 1735; 1H NMR (400 MHz, CDCl3) δ = 1.31 (t, 3H, J = 7.4 Hz, CH3 SEt), 2.71 (m, 2H, CH2 SEt), 3.64 (s, 3H, CH3 COOMe), 3.84 (dd, 1H, J = 7.0 Hz, J = 3.0 Hz, H-3), 4.00 (dd, 1H, J = 5.2, J = 3.0 Hz, H-2), 4.16 (t, 1H, J = 7.0 Hz, J = 6.2 Hz, H-4), 4.56 (d, 1H, J = 6.2 Hz, H-5), 4.61 (s, 2H, CH2Ph), 4.64 (d, 1H, J = 11.6 Hz, CHHPh), 4.68 (d, 1H, J = 11.6 Hz, CHHPh), 5.35 (d, 1H, J = 5.2 Hz, H-1); 7.24 – 7.35 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 14.9 (CH3 SEt), 25.5 (CH2 SEt), 52.2 (CH3 COOMe), 68.3 (C-2), 71.8 (C-5), 72.5 (CH2 Bn), 73.7 (CH2 Bn), 75.4 (C-4), 78.6 (C-3), 84.5 (C-1), 127.7 – 128.5 (CH Arom), 137.3 (Cq Bn), 137.7 (Cq Bn), 169.7 (C=O); HRMS: [M+NH4]+ calcd for C23H32O6SN 450.19503, found 450.19748. Ethyl 2-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranosidurono-6,3-lactone (13): Diol 12 was converted according to the general procedure delivering lactone 13 in 72% yield as a colorless oil. TLC: 20% EtOAc/PE; [α]D22: +40° (c = 0.5, CHCl3); IR (neat, cm-1): 948, 1012, 1058, 1161, 1224, 1373, 1456, 1741, 1799, 2873; 1H NMR (400 MHz, CDCl3) δ = 1.25 (t, 3H, J = 7.2 Hz,

O O

SEt OAc

O OBn

CH3 SEt), 2.13 (s, 3H, CH3 Ac), 2.71 (m, 2H, CH2 SEt), 4.07 (dd, 1H, J = 5.9 Hz, J = 2.8 Hz, H-4), 4.28 (dd, 1H, J = 2.8 Hz, J = 1.0 Hz, H-5), 4.63 (d, 1H, J = 11.8 Hz, CHHPh), 4.69 (d, 1H, J = 9.2 Hz, H-1), 4.75 (d, 1H, J = 11.8 Hz, CHHPh), 4.79 (dt, 1H, J = 5.9 Hz, J = 1.2 Hz, J = 1.0 Hz, H-3), 5.40 (dd, 1H, J = 1.2 Hz, H-2), 7.26 – 7.40 (m, 5H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 14.8 (CH3 SEt), 20.8 (CH3 Ac), 23.9 (CH2 SEt), 67.3 (C-2), 72.0 (CH2 Bn), 72.1 (C-5), 74.8 (C-4), 77.5 (C-3), 80.3 (C-1), 128.1 – 128.7 (CH Arom), 136.0 (Cq Bn), 169.5 (C=O Ac or lactone), 169.6 (C=O lactone or Ac); HRMS: [M+NH4]+ calcd for C17H24O6SN 370.13243, found 370.12914. MeOOC BnO HO

Methyl (ethyl 2-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranoside) uronate (14): Compound 14 was obtained in an analogous way as described for compound 11. TLC: 20% EtOAc/PE;

OAc O

[α]D22: +35° (c = 0.6, CHCl3); IR (neat, cm-1): 1035, 1091, 1232, 1373, 1438, 1739, 2858; 1H NMR (400 MHz, CDCl3) δ = 1.30 (t, 1H, J = 7.6 Hz, CH3 SEt), 2.16 (s, 3H, CH3 Ac), 2.65 (m, 2H, CH2 SEt), 3.78 (s, 3H, CH3 COOMe), 3.98 (t, 1H, J = 8.8 Hz, H-4), 4.08 (dd, 1H, J = 8.8 Hz, J = 8.4 Hz, J = 3.3 Hz, H-3), 4.58 (d, 1H, J = 8.4 Hz, H-5), 4.68 (d, 1H, J = 11.2 Hz, CHHPh), 4.72 (d, 1H, J = 11.2 Hz, CHHPh), 5.20 (t, 1H, J = 3.3, J = 2.8 Hz, H-2), 5.40 (d, 1H, J = 2.8 Hz, H-1), 7.26 – 7.37 (m, 5H, H Arom); 13C NMR (100 MHz, SEt

CDCl3) δ = 14.8 (CH3 SEt), 22.4 (CH3 Ac), 25.1 (CH2 SEt), 52.5 (CH3 COOMe), 70.1 (C-3), 71.3 (C-5), 72.8 (C-2), 74.5 (CH2 Bn), 77.4 (C-4), 81.9 (C-1), 127.8 – 128.5 (CH Arom), 137.8 (Cq Bn), 169.8 (C=O Ac or lactone), 170.5 (C=O lactone or Ac); HRMS: [M+NH4]+ calcd for C18H28O7SN 402.15865, found 402.16217. O SPh

O O BnO

58   

58

OBn

Phenyl 2,4-di-O-benzyl-1-thio-β-D-glucopyranosidurono-6,3-lactone (16): Diol 15 was converted according to the general procedure delivering lactone 16 in 51% yield as a colorless oil. TLC: 20% EtOAc/PE; [α]D22: -28° (c = 0.4, CDCl3); IR (neat, cm-1): 999, 1024, 1078, 1367, 1454, 1585,

1‐Thio Uronic Acid Lactones in Oligosaccharide Synthesis    1637, 1801, 3006, 3223; H NMR (400 MHz, CDCl3) δ = 3.83 (d, 1H, J = 5.4 Hz, H-2), 4.06 (t, 1H, J = 3.2 Hz, J = 2.4 Hz, J = 1.0 Hz, H-4), 4.10 (d, 1H, J = 2.4 Hz, H-5), 4.56 (d, 1H, J = 11.6 Hz, CHHPh), 4.60 (d, 1H, J = 11.6 Hz, CHHPh), 4.64 (dd, 1H, J = 3.2 Hz, J = 1.0 Hz, H-3), 4.68 (d, 1H, J = 11.6 Hz, CHHPh), 4.70 (d, 1H, J = 11.6 Hz, CHHPh), 5.61 (d, 1H, J = 5.4 Hz, H-1), 7.19 – 7.43 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) 1

δ = 69.7 (C-5), 72.2 (CH2 Bn), 73.4 (CH2 Bn), 74.5 (C-4), 74.7 (C-3), 76.9 (C-2), 84.4 (C-1), 128.0 – 133.2 (CH Arom), 132.3 (Cq SPh), 136.3 (Cq Bn), 137.0 (Cq Bn), 170.5 (C=O); HRMS: [M+NH4]+ calcd for C26H28O5SN 466.16882, found 466.17197.

BnO HO

COOMe O

Methyl (phenyl 2,4-di-O-benzyl-1-thio-β-D-glucopyranoside) uronate (17): Compound 17

was obtained in an analogous way as described for compound 8. TLC: 20% EtOAc/PE; IR OBn (neat, cm-1): 1026, 1074, 1207, 1438, 1747, 2920; 1H NMR (400 MHz, CDCl3) δ = 3.92 (dd, 1H, J = 9.6 Hz, J = 8.8 Hz, H-2), 3.72 (t, 1H, J = 9.2 Hz, J = 8.8 Hz, H-4), 3.75 (s, 3H, CH3 COOMe), 3.78 (t, 1H, J = 9.2 Hz, J = 8.8 Hz, H-3), 3.91 (d, 1H, J = 9.2 Hz, H-5), 4.63 (d, 1H, J = 11.2 Hz, CHHPh), 4.66 (d, 1H, J = 9.6 Hz, H-1), 4.68 (d, 1H, J = 10.4 Hz, CHHPh), 4.76 (d, 1H, J = 11.2 Hz, CHHPh), 4.96 (d, 1H, J = SPh

10.4 Hz, CHHPh), 7.25 – 7.55 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.5 (CH3 COOMe), 74.8 (CH2 Bn), 75.2 (CH2 Bn), 77.6 (C-5), 77.7 (C-3), 78.6 (C-4), 80.0 (C-2), 88.0 (C-1), 127.8 – 132.0 (CH Arom), 137.4 (2xCq Bn), 170.9 (C=O COOMe); HRMS: [M+NH4]+ calcd for C27H32O6SN 498.19503, found 498.19424. 2,3,4-Tri-O-benzyl-β-D-glucopyranosidurono-6,1-lactone (19): Diol 18 was converted according to the general procedure delivering lactone 19 in 27% yield as a colorless oil. TLC: 20% EtOAc/PE; [α]D22: -5° (c = 0.2, CHCl3); IR (neat, cm-1): 912, 1043, 1068, 1178, 1454, 1807, 2866, BnO OBn 3340; 1H NMR (400 MHz, CDCl3) δ = 3.50 (s, 1H, H-2), 3.63 (s, 1H, H-4 or H-3), 3.68 (t, 1H, J = 1.6 Hz, H-3 or H-4), 4.26 (d, 1H, J = 12.4 Hz, CHHPh), 4.40 (d, 1H, J = 12.4 Hz, CHHPh), 4.47 (d, 1H, J = 12.4 O

OBn O

O

Hz, CHHPh), 4.54 (d, 1H, J = 12.4 Hz, CHHPh), 4.59 (d, 1H, J = 12.4 Hz, CHHPh), 4.64 (d, 1H, J = 12.4 Hz, CHHPh), 5.79 (s, 1H, H-1), 7.15 – 7.36 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 71.6 (CH2 Bn), 72.0 (CH2 Bn), 72.1 (C-5), 72.4 (C-4 or C-3), 72.4 (CH2 Bn), 73.0 (C-2), 75.0 (C-3 or C-4), 102.5 (C-1), 127.7 – 128.6 (CH Arom), 136.9 (Cq Bn), 170.0 (C=O); HRMS: [M+Na]+ calcd for C27H26O6Na 469.16216, found 469.15979. O O

O BnO

O

O BnO OBn OBn

OMe OBn

Methyl 2,3,4-tri-O-benzyl-6-O-(2,4-di-O-benzyl-α-D-galactopyranosyl-urono6,3-lactone)-α-D-glucopyranoside (21): Donor 7 was glycosylated with acceptor 20 in the same way as described in the general procedure delivering the title compound 21 as a colorless oil. TLC: 30% EtOAc/PE; IR (neat, cm-1):

694, 734, 979, 1026, 1141, 1203, 1355, 1436, 1759; 1H NMR (400 MHz, CDCl3) δ = 3.22 (s, 3H, CH3 OMe), 3.42 (dd, 1H, J = 10.0 Hz, J = 3.5 Hz, H-2), 3.54 (t, 1H, J = 9.5 Hz, H-4), 3.68 (d, 1H, J = 11.0 Hz, H-6), 3.73 (d, 1H, J = 9.5 Hz, H-5), 3.97 (t, 1H, J = 9.5 Hz, H-3), 4.00 (bs, 1H, H-2’), 4.11 (s, 1H, H-4’), 4.19 (dd, 1H, J = 11.0 Hz, J = 3.5 Hz, H-6), 4.44 (s, 1H, H-5’), 4.54 (s, 1H, H-1), 4.55 (s, 2H, CH2Ph), 4.57 (d, 1H, J = 12.5 Hz, CHHPh), 4.62 (d, 1H, J = 12.0 Hz, CHHPh), 4.65 (d, 1H, J = 10.0 Hz, CHHPh), 4.69 (d, 1H, J = 5.0 Hz, H-3’), 4.76 (d, 1H, J = 10.5 Hz, CHHPh), 4.79 (d, 1H, J = 12.0 Hz, CHHPh), 4.83 (d, 1H, J = 11.0 Hz, CHHPh), 4.89 (s, 1H, H-1’), 4.90 (d, 1H, J = 12.5 Hz, CHHPh), 4.96 (d, 1H, J = 11.0 Hz, CHHPh), 7.24 – 7.65 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 55.1 (CH3 OMe), 68.5 (C-6), 69.8 (C-5), 71.6 (CH2 Bn), 73.3 (CH2 Bn), 74.2 (CH2 Bn), 74.4 (C-2’), 75.1 (CH2 Bn), 76.0 (C-5’), 77.7 (C-4), 79.9 (C-2), 80.2 (C-3’), 81.7 (C-3), 98.0 (C-1), 98.8 (C-1’), 124.7 – 130.9 (CH Arom), 136.7 (Cq Bn), 137.5 (Cq Bn), 138.0 (Cq Bn), 138.1 (Cq Bn), 138.7 (Cq Bn), 171.6 (C=O lactone); 13C-GATED (125 MHz, CDCl3): 98.0 (JC1,H1 = 161 Hz, C-1), 102.4 (JC1’,H1’ = 163 Hz, C-1’); ESI-MS: 825.3 [M+Na]+.

59   

59

Chapter 3    O O

O O

BnO

OMe OAc

O AcO OAc OBn

Methyl 2,3,4-tri-O-acetyl-6-O-(2,4-di-O-benzyl-α-D-galactopyranosylurono6,3-lactone)-α-D-glucopyranoside (23): Donor 7 was glycosylated with acceptor 22 in the same way as described in the general procedure delivering the title compound 23 as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: +82°

(c = 1, CHCl3); IR (neat, cm-1): 925, 1028, 1159, 1217, 1367, 1454, 1747, 1799, 2920; 1H NMR (400 MHz, CDCl3) δ = 1.94 (s, 6H, 2xCH3 Ac), 2.00 (s, 3H, CH3 Ac), 3.28 (s, 3H, CH3 OMe), 3.62 (d, 1H, J = 11.6 Hz, H6), 3.84 (bd, 1H, J = 10.4 Hz, H-5), 3.89 – 3.94 (m, 2H, H-6 and H-2’), 4.05 (s, 1H, H-4’), 4.44 (s, 1H, H-5’), 4.51 (s, 2H, CH2Ph), 4.53 (d, 1H, J = 12.0 Hz, CHHPh), 4.62 (d, 1H, J = 4.0 Hz, H-3’), 4.74 (dd, 1H, J = 10.2 Hz, J = 3.6 Hz, H-2), 4.81 (s, 1H, H-1’), 4.83 (d, 1H, J = 3.2 Hz, H-1), 4.86 (d, 1H, J = 12.0 Hz, CHHPh), 5.04 (t, 1H, J = 9.6 Hz, H-4), 5.38 (t, 1H, J = 10.2 Hz, J = 9.6 Hz, H-3), 7.19 – 7.48 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.6 (3xCH3 Ac), 55.4 (CH3 OMe), 67.8 (C-6), 68.3 (C-4), 68.6 (C-5), 70.4 (C-3), 70.7 (C-2), 71.6 (CH2 Bn), 72.0 (C-4’), 74.2 (CH2 Bn), 74.3 (C-2’), 75.5 (C-5’), 80.3 (C-3’), 96.7 (C-1), 98.9 (C-1’), 127.8 – 128.6 (CH Arom), 136.8 (Cq Bn), 137.5 (Cq Bn), 169.3 (C=O Ac or lactone), 170.1 (C=O Ac or lactone), 170.1 (C=O Ac or lactone), 171.7 (C=O Ac or lactone); 13C-GATED (100 MHz, CDCl3): 96.7 (JC1,H1 = 169 Hz, C-1), 98.9 (JC1’,H1’ = 158 Hz, C-1’); HRMS: [M+NH4]+ calcd for C33H42O14N 676.26053, found 676.26440. O O O BnO

O

COOMe O

Methyl (methyl 2,3-di-O-benzyl-4-O-(2,4-di-O-benzyl-α-D-galactopyranosylurono-6,3-lactone)-α-D-glucopyranoside) uronate (25): Donor 7 was glycosylated

with acceptor 24 in the same way as described in the general procedure delivering the title compound 25 as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: +6° (c = 0.5, CHCl3); IR (neat, cm-1): 915, 1030, 1057, 1080, 1219, 1360, 1450, 1504, 1730, 1809, 2925; 1H NMR (400 MHz, CDCl3) δ = 3.39 (s, 3H, CH3 OMe), 3.53 (dd, 1H, J = 5.0 Hz, J = 2.4 Hz, H-2’), 3.56 (dd, 1H, J = 6.5 Hz, J = 3.2 Hz, H-2), 3.67 (s, 3H, CH3 COOMe), 3.87 (m, 1H, J = 2.5 Hz, H-3, H-5), 4.00 (s, 1H, H-4’), 4.15 (dd, BnO

OBn

BnO

OMe

1H, J = 7.3 Hz, J = 2.5 Hz, H-4), 4.30 (d, 1H, J = 11.9 Hz, CHHPh), 4.34 (s, 1H, H-5’), 4.51 (s, 2H, CH2 Bn), 4.54 (dd, 1H, J = 5.0 Hz, J = 1.6 Hz, H-3’), 4.59 (d, 1H, J = 11.7 Hz, CHHPh), 4.60 (s, 1H, H-1), 4.61 (d, 1H, J = 12.0 Hz, CHHPh), 4.72 (d, 1H, J = 11.9 Hz, CHHPh), 4.75 (d, 1H, J = 12.0 Hz, CHHPh), 4.99 (d, 1H, J = 11.7 Hz, CHHPh), 5.03 (d, 1H, J = 2.4 Hz, H-1’), 7.18 – 7.35 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.3 (CH3 COOMe), 55.8 (CH3 OMe), 70.3 (C-4’), 71.3 (CH2 Bn), 71.9 (C-4), 74.2 (CH2 Bn), 74.3 (C-2’), 74.4 (CH2 Bn), 74.5 (CH2 Bn), 75.4 (C-5’), 78.2 (C-5 or C-3), 79.2 (C-2), 80.4 (C-3 or C-5), 98.4 (C-1), 99.1 (C-1’), 127.3 – 128.6 (CH Arom), 137.6 (Cq Bn), 138.6 (Cq Bn), 168.9 (C=O COOMe), 172.6 (C=O lactone); 13CGATED (100 MHz, CDCl3): 98.4 (JC1,H1 = 169 Hz, C-1), 99.1 (JC1’,H1’ = 164 Hz, C-1’); HRMS: [M+NH4]+ calcd for C48H52O13N 850.34387, found 850.34180. Methyl (2-(N-benzyloxycarbonyl)-amino-3,6-di-O-benzyl-2-deoxy-4-O-(2,4-di(27): O-benzyl-α-D-galactopyranosylurono-6,3-lactone)-α-D-glucopyranoside O O O BnO Donor 7 was glycosylated with acceptor 26 in the same way as described in the BnO CbzHN OBn OMe general procedure delivering the title compound 27 as a colorless oil. N.B.: The amount of TTBP added to the reaction mixture was 1 equiv. with respect to the amount of Tf2O. TLC: 30% O

O

OBn

EtOAc/PE; [α]D22: +31° (c = 1, CHCl3); IR (neat, cm-1): 912, 1028, 1102, 1153, 1211, 1356, 1401, 1724, 1753; H NMR (400 MHz, CDCl3) δ = 3.36 (s, 3H, CH3 OMe), 3.47 (d, 1H, J = 2.8 Hz, H-2’), 3.61 (t, 1H, J = 8.8 Hz, H-3), 3.63 – 3.75 (m, 2H, H-5 and H-6), 3.78 (t, 1H, J = 8.8 Hz, H-4), 3.89 (d, 1H, J = 10.8 Hz. H-6), 3.99 (s, 1H, H-4’), 4.12 (t, 1H, J = 10.0 Hz, J = 8.0 Hz, H-2), 4.28 (s, 1H, H-5’), 4.37 (d, 1H, J = 12.0 Hz, CHHPh), 4.44 1

(d, 1H, J = 11.6 Hz, CHHPh), 4.49 (s, 1H, H-3’), 4.51 (d, 1H, J = 10.8 Hz, CHHPh), 4.60 (d, 1H, J = 11.6 Hz, CHHPh), 4.65 (d, 1H, J = 12.0 Hz, CHHPh), 4.67 (s, 1H, H-1), 4.72 (d, 1H, J = 10.8 Hz, CHHPh), 5.00 (d, 1H, J = 10.0 Hz, NH), 5.07 (d, 1H, J = 10.0 Hz, CHHPh), 5.10 (s, 1H, H-1’), 5.11 (d, 1H, J = 10.0 Hz, CHHPh), 7.16

60   

60

1‐Thio Uronic Acid Lactones in Oligosaccharide Synthesis    – 7.65 (m, 25H, H Arom); C NMR (100 MHz, CDCl3) δ = 54.6 (C-2), 55.0 (CH3 OMe), 66.8 (C-6), 69.9 (C-4), 71.2 (CH2 Cbz), 71.8 (C-4’), 73.4 (CH2 Bn), 74.4 (CH2 Bn), 74.8 (C-5’), 75.6 (C-2’), 79.6 (C-3’), 81.4 (C-3), 98.9 (C-1), 101.3 (C-1’), 126.8 – 130.9 (CH Arom), 136.7 (Cq Bn), 137.7 (Cq Bn), 137.8 (Cq Bn), 138.3 (Cq Bn), 155.6 (C=O Cbz), 171.1 (C=O lactone); HRMS: [M+Na]+ calcd for C49H55N2O12 868.33035, found 868.33606. 13

OpMP O

O MeOOC O

O BnO

O

OBn

BnO

Methyl (para-methoxyphenyl 2,3-di-O-benzyl-4-O-(2,4-di-O-benzyl-α/β-Dgalactopyranosylurono-6,3-lactone)-α-D-galactopyranoside) uronate (29): Donor 7 was glycosylated with acceptor 28 in the same way as described in the general procedure delivering title compound 29 as a colorless oil. The anomers were

separated with flash chromatography. TLC: 30% EtOAc/PE; α-anomer: [α]D22: -30° (c = 1, CHCl3); IR (neat, cm-1): 910, 1028, 1058, 1080, 1217, 1363, 1454, 1506, 1735, 1809, 2922; 1H NMR (400 MHz, CDCl3) δ = 3.58 (dd, 1H, J = 8.8 Hz, J = 4.6 Hz, J = 3.2 Hz, H-3), 3.74 (s, 3H, CH3 OMe or COOMe), 3.75 (s, 3H, CH3 COOMe or OMe), 3.91 (dd, 1H, J = 8.8 Hz, J = 1.6 Hz, H-2), 3.93 (s, 1H, H-2’), 4.05 (s, 1H, H-5), 4.12 (s, 1H, H-4’), 4.50 (d, 1H, J = 11.6 Hz, CHHPh), 4.54 (s, 1H, H-4), 4.56 (d, 1H, J = 11.6 BnO

Hz, CHHPh), 4.58 (s, 1H, H-5’), 4.62 (d, 1H, J = 12.0 Hz, CHHPh), 4.67 (d, 1H, J = 11.6 Hz, CHHPh), 4.70 (s, 1H, H-3’), 4.78 (d, 1H, J = 10.8 Hz, CHHPh), 4.80 (s, 1H, H-1’), 4.81 (d, 1H, J = 8.8 Hz, H-1), 4.85 (d, 1H, J = 12.0 Hz, CHHPh), 4.86 (d, 1H, J = 11.6 Hz, CHHPh), 5.02 (d, 1H, J = 10.8 Hz, CHHPh), 6.78 (d, 2H, J = 9.0 Hz, 2xH pMP Arom), 7.03 (d, 2H, J = 9.0 Hz, 2xH pMP Arom), 7.23 – 7.42 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 51.7 (CH3 OMe or COOMe), 53.9 (CH3 COOMe or OMe), 69.6 (CH2 Bn), 71.3 (C-4), 71.6 (C-5), 74.2 (CH2 Bn), 74.6 (C-2’), 75.6 (CH2 Bn), 75.7 (C-5’), 75.8 (C-4), 78.0 (C-2), 78.8 (C-3), 80.4 (C-3’), 99.1 (C-1’), 102.5 (C-1), 107.4 (CH pMP Arom), 119.6 (CH pMP Arom), 123.8 – 124.4 (CH Arom), 137.8 (Cq Bn), 137.9 (Cq Bn), 138.4 (Cq Bn), 138.8 (Cq Bn), 151.7 (Cq pMP), 155.6 (Cq pMP), 167.9 (C=O lactone), 173.7 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 99.1 (JC1’,H1’ = 160 Hz, C-1’), 102.5 (JC1,H1 = 159 Hz, C-1); HRMS: [M+NH4]+ calcd for C48H48O13 850.34387, found 850.34180. β-anomer: [α]D22: +15° (c = 1, CHCl3); IR (neat, cm-1): 910, 1028, 1058, 1080, 1217, 1363, 1454, 1506, 1735, 1809, 2922; 1H NMR (400 MHz, CDCl3) δ = 3.67 (dd, 1H, J = 9.8 Hz, J = 2.4 Hz, H-3), 3.82 (s, 3H, CH3 COOMe or OMe), 3.97 (s, 3H, CH3 OMe or COOMe), 3.98 (dd, 1H, J = 7.6 Hz, J = 2.4 Hz, H-2), 4.04 (s, 1H, H-4’), 4.08 (s, 1H, H-5’), 4.11 (d, 1H, J = 4.8 Hz, H-2’), 4.23 (d, 1H, J = 12.0 Hz, CHHPh), 4.34 (s, 1H, H-5’), 4.39 (d, 1H, J = 12.0 Hz, CHHPh), 4.61 (s, 2H, CH2 Bn), 4.66 (s, 1H, H-4), 4.68 (d, 1H, J = 4.8 Hz, H-3’), 4.81 (s, 2H, CH2 Bn), 4.86 (d, 1H, J = 7.6 Hz, H-1), 4.90 (d, 1H, J = 10.8 Hz, CHHPh), 5.07 (d, 1H, J = 10.8 Hz, CHHPh), 5.60 (s, 1H, H-1’), 6.87 (d, 2H, J = 9.2 Hz, 2xH pMP Arom), 7.15 (d, 2H, J = 9.2 Hz, 2xH pMP Arom), 7.30 – 7.43 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.9 (CH3 OMe or COOMe), 55.6 (CH3 COOMe or OMe), 70.7 (C-4’), 71.2 (CH2 Bn), 72.2 (CH2 Bn), 73.3 (C-5), 73.5 (C-4), 74.0 (CH2 Bn), 75.1 (CH2 Bn), 75.9 (C-5’), 77.5 (C-2), 78.1 (C-2’), 78.5 (C3’), 81.5 (C-3), 100.5 (C-1’), 103.5 (C-1), 114.4 (CH pMP Arom), 119.08 (CH pMP Arom), 127.6 – 128.6 (CH Arom), 136.8 (Cq Bn), 137.1 (Cq Bn), 137.3 (Cq Bn), 138.1 (Cq Bn), 151.4 (Cq pMP), 155.5 (Cq pMP), 166.9 (C=O lactone), 172.0 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 100.5 (JC1’,H1’ = 174 Hz, C-1’), 103.5 (JC1,H1 = 159 Hz, C-1); HRMS: [M+NH4]+ calcd for C48H52O13N 850.34387, found 850.33893. BnO HO

COOMe O BnO

O BnO

OBn O CbzHN

Methyl (2-(N-benzyloxycarbonyl)-amino-3,6-di-O-benzyl-2-deoxy-4-O-(methyl 2,4di-O-benzyl-α-D-galactopyranosyluronate)-α-D-glucopyranoside (30): Lactone 27 was dissolved in anhydrous methanol before a catalytic amount KOtBu (10 mol%) was added. Stirring was continued for 4h before the mixture was neutralized with

OMe

Amberlite IR-120 H+-resin. After filtration and purification by flash chromatography title compound 30 was isolated as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: +94° (c = 1, CHCl3); IR (neat, cm-1): 912, 1028, 1102, 1153, 1211, 1356, 1401, 1724, 1753; 1H NMR (400 MHz, CDCl3) δ = 3.33 (s, 3H, CH3

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Chapter 3    OMe), 3.43 (s, 3H, CH3 COOMe), 3.61 (d, 1H, J = 9.6 Hz, H-6), 3.77 – 3.80 (m, 3H, H-4, H-5 and H-2’), 3.85 (d, 1H, J = 9.6 Hz, H-6), 4.01 (dd, 1H, J = 10.0 Hz, J = 3.0 Hz, H-3’), 4.12 – 4.16 (m, 2H, H-2 and H-4’), 4.18 (t, 1H, J = 9.6 Hz, H-3), 4.34 (d, 1H, J = 12.0 Hz, CHHPh), 4.46 (s, 1H, H-5’), 4.52 (d, 1H, J = 11.6 Hz, CHHPh), 4.55 (d, 1H, J = 12.0 Hz, CHHPh), 4.56 (s, 2H, CH2 Bn), 4.58 (d, 1H, J = 11.6 Hz, CHHPh), 4.67 (d, 1H, J = 11.2 Hz, CHHPh), 4.70 (s, 1H, H-1), 4.72 (d, 1H, J = 11.2 Hz, CHHPh), 4.94 (d, 1H, J = 10.0 Hz, NH), 4.98 (d, 1H, J = 12.4 Hz, CHHPh), 5.03 (d, 1H, J = 12.4 Hz, CHHPh), 5.77 (d, 1H, J = 3.2 Hz, H-1’), 7.18 – 7.34 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.0 (CH3 COOMe), 53.9 (C-2), 55.1 (CH3 OMe), 66.9 (CH2 Bn), 68.8 (CH2 Bn), 69.4 (C-3’), 70.2 (C-4, C-5 or C-2’), 70.9 (C-5’), 72.1 (CH2 Cbz), 72.3 (C-4’), 72.8 (CH2 Bn), 73.3 (CH2 Bn), 74.9 (CH2 Bn), 75.6 (C-4, C-5 or C-2’), 77.8 (C-3), 80.7 (C-4, C-5 or C-2’), 96.6 (C-1’), 98.7 (C-1), 126.8 – 128.4 (CH Arom), 137.5 (Cq Bn), 137.9 (Cq Bn), 138.0 (Cq Bn), 155.7 (C=O Cbz), 168.9 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 96.6 (JC1’,H1’ = 170 Hz, C-1’), 98.7 (JC1,H1 = 172 Hz, C1); HRMS: [M+Na]+ calcd for C50H55NO13Na 900.35656, found 900.35333. AcO TBSO

OBz O OBz

BnO O

COOMe O BnO

O BnO

OBn O CbzHN

OMe

Methyl (2-(N-benzyloxycarbonyl)-amino-3.6-di-O-benzyl-2-deoxy-4-O-(methyl 2,4-di-O-benzyl-3-O-(4-Oacetyl-2,6-di-O-benzoyl-3-O-tert-butyldimethylsilyl-β-D-galactopyranosyl)-α-D-galactopyranosyluronate)-α-Dglucopyranoside (32): A solution of 63 mg donor 31 (0.1 mmol) and 26 mg diphenylsulfoxide (0.13 mmol, 1.3 equiv.) in 3 mL DCM was stirred over activated MS3Å for 30 min. The mixture was cooled to -60°C before 21 µL triflic acid anhydride (0.13 mmol, 1.3 equiv.) was added. The mixture was allowed to warm to -40°C in 1h followed by addition of 87 mg acceptor 30 (0.1 mmol, 1.0 equiv.) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to -15°C. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded 88 mg of the title compound 32 (0.063 mmol, 63%) as a light yellow oil. TLC: 30% EtOAc/PE; [α]D22: +35° (c = 1, CHCl3); IR (neat, cm-1): 912, 1102, 1153, 1211, 1401, 1724, 1753; 1H NMR (600 MHz, CDCl3) δ = -0.13 (s, 3H, CH3 TBDMS), -0.07 (s, 3H, CH3 TBDMS), 0.69 (s, 9H, tBu TBDMS), 2.19 (s, 3H, CH3 Ac), 3.31 (s, 3H, CH3 COOMe), 3.34 (s, 3H, CH3 OMe), 3.51 (d, 1H, J = 10.9 Hz, H-6), 3.60 (t, 2H, J = 10.2 Hz, J = 9.3 Hz, H-4 and H-5), 3.73 (d, 1H, J = 11.0 Hz, H-6), 3.83 (dd, 1H, J = 8.6 Hz, J = 3.1 Hz, H-2’), 3.91 (m, 2H, H-3’’ and H-5’’), 4.02 (t, 1H, J = 9.3 Hz, H-2), 4.07 (t, 1H, J = 9.3 Hz, H-3), 4.10 (d, 1H, J = 12.8 Hz, CHHPh), 4.21 (dd, 1H, J = 10.3 Hz, J = 2.6 Hz, H-3’), 4.30 (d, 1H, J = 12.8 Hz, CHHPh), 4.32 (s, 1H, H-4’), 4.36 (s, 1H, H5’), 4.40 (dd, 1H, J = 11.2 Hz, J = 5.5 Hz, H-6’’), 4.41 (d, 1H, J = 12.1 Hz, CHHPh), 4.43 (d, 1H, J = 12.1 Hz, CHHPh), 4.46 (d, 1H, J = 12.1 Hz, CHHPh), 4.52 (dd, 1H, J = 11.2 Hz, J = 7.0 Hz, H-6’’), 4.63 (d, 1H, J = 11.8 Hz, CHHPh), 4.65 (d, 1H, J = 12.1 Hz, CHHPh), 4.66 (s, 1H, H-1), 4.77 (d, 1H, J = 9.9 Hz, NH), 4.89 (d, 1H, J = 8.0 Hz, H-1’’), 4.95 (s, 2H, CH2 Bn), 4.99 (d, 1H, J = 11.8 Hz, CHHPh), 4.31 (d, 1H, J = 3.1 Hz, H-1’), 5.40 (s, 1H, H-4’’), 5.54 (t, 1H, J = 8.1 Hz, H-2’’), 6.92 – 8.08 (m, 35H, H Arom); 13C NMR (150 MHz, CDCl3) δ = -4.8 (2xCH3 TBDMS), 17.6 (Cq tBu TBDMS), 20.9 (CH3 Ac), 25.2 (tBu TBDMS), 51.8 (CH3 OMe), 54.0 (C-2), 55.1 (CH3 COOMe), 62.4 (C-6’’), 66.8 (CH2 Bn), 68.6 (C-6), 70.2 (C-4’’), 70.5 (C-5), 71.0 (C-3’’ and C-5’’), 71.3 (C-3’), 72.6 (C-2’’), 72.8 (CH2 Cbz), 73.1 (CH2 Bn), 73.5 (CH2 Bn), 74.3 (C-3), 74.7 (CH2 Bn), 75.1 (C2’), 75.2 (C-3), 77.8 (C-3’’), 80.0 (C-4), 97.8 (C-1’), 98.6 (C-1), 101.7 (C-1’’), 127.1 – 133.3 (CH Arom), 128.6 (Cq Bz), 129.5 (Cq Bz), 133.2 (Cq Bn), 138.2 (Cq Bn), 138.4 (Cq Bn), 138.5 (Cq Bn), 155.7 (C=O Cbz), 165.0 (C=O Bz, Ac or COOMe), 166.0 (C=O Bz, Ac or COOMe), 168.6 (C=O Bz, Ac or COOMe), 170.2 (C=O Bz,

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Ac or COOMe); C-GATED (150 MHz, CDCl3): 97.8 (JC1’,H1’ = 172 Hz, C-1’), 98.6 (JC1,H1 = 169 Hz, C-1), 101.7 (JC1’’,H1’’ = 159 Hz, C-1’’); HRMS: [M+Na]+ calcd for C78H89NO21SiNa 1426.52575, found 1426.53050.

References and Notes 1

Original publication: Van den Bos, L.J.; Litjens, R.E.J.N.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2005, 7, 2007–2010.

2 3

See Chapter 1. (a) Cella, J.A.; Kelley, J.A.; Kenhan, E.F. J. Org. Chem. 1975, 40, 1860–1862 (b) Ganem, B. J. Org. Chem. 1975, 40, 1998–2000 (c) Cella, J.A.; McGrath, J.P.; Kelley, J.A.; El Soukkary, O.; Hilpert, L. J. Org. Chem. 1977, 42, 2077–2080. (a) Anelli, P.L.; Biffi, C.; Montanari, F.; Quici, S. J. Org. Chem. 1987, 52, 2559–2562 (b) Anelli, P.L.;

4

Banfi, S.; Montanari, F.; Quici, S. J. Org. Chem. 1989, 54, 2970–2972 (c) Siedlecka, R.; Skarzewski, J.; Mlochowski, J. Tetrahedron Lett. 1990, 31, 2177–2180 (d) Davis, N.J.; Flitsch, S.L. Tetrahedron Lett. 1993, 34, 1181–1184 (e) De Nooy, A.E.J.; Besemer, A.C.; Van Bekkum, H. Carbohydr. Res. 1995, 269, 89–98 (f) Haller, M.; Boons, G.-J. J. Chem. Soc., Perkin Trans. 1 2001, 814–822 (g) Litjens, R.E.J.N.; Den Heeten, R.; 5

6 7 8

Timmer, M.S.M.; Overkleeft, H.S.; Van der Marel, G.A. Chem. Eur. J. 2005, 11, 1010–1016. (a) Spyroudis, S.; Varvoglis, A. Synthesis 1975, 445–447 (b) Narasaka, K.; Morikawa, A.; Saigo, K.; Mukaiyama, T. Bull. Chem. Soc. Jpn. 1977, 50, 2773–2776 (c) Muller, P.; Godoy, J. Tetrahedron Lett. 1981, 22, 2361–2364 (d) Muller, P.; Godoy, J.; Helv. Chim. Acta 1983, 66, 1790–1795 (e) Togo, H.; Aoki, M.; Kuramochi, T.; Yokoyama, M. J. Chem. Soc., Perkin Trans. 1 1993, 2417–2427 (f) Magnus, P.; Lacour, J.; Evans, P.A.; Roe, M.B.; Hulme, C. J. Am. Chem. Soc. 1996, 118, 3406–3418. De Mico, A.; Margarita, R.; Parlanti, L.; Vescovi, A.; Piancatelli, G. J. Org. Chem. 1997, 62, 6974–6977. For oxidation towards the carboxylic acid: Epp, J.B.; Widlanski, T.S. J. Org. Chem. 1999, 64, 293–295. Hansen, T.M.; Florence, G.J.; Lugo-Mas, P.; Chen, J.H.; Abrams, J.N.; Forsyth, C.J. Tetrahedron Lett. 2003, 44, 57–59.

9

For a comprehensive review on the mechanism of TEMPO-oxidations see: De Nooy, A.E.J.; Besemer, A.C.; Van Bekkum, H. Synthesis 1996, 1153–1174. 10 (a) see Chapter 2 (b) Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168. 11 For a review on the oxidation of carbohydrates using TEMPO see: Bragd, P.L.; Van Bekkum, H.; Besemer A.C. Top. Catal. 2004, 27, 49–66. 12 See Chapter 7 for the method of synthesis. 13 Formation of the lactone ring was confirmed by the change in coupling constants (3J1,2) of compounds 6 and 7. The doublet in compound 6 (3J1,2 = 7.1 Hz) was transposed into a singlet after the oxidationlactonization process. 14 (a) Chernyak, A.Y.; Kononov, L.O.; Kochetkov, N.K. Carbohydr. Res. 1992, 216, 381–398 (b) Kornilov, A.V.; Sherman, A.A.; Kononov, L.O.; Shashkov, A.S.; Nifant'ev, N.E. Carbohydr. Res. 2000, 329, 717–730. 15 The reason for this is somewhat unclear, but it is most probably due to steric hindrance of the bulky benzyl and thiophenyl groups (cf lactones 10 and 13). 16 For a related study on the use of 6,1-lactones see: Poláková, M.; Pitt, N.; Tosin, M.; Murphy, P.V. Angew. Chem. Int. Ed. 2004, 116, 2572–2575. 17 Litjens, R.E.J.N.; Van den Bos, L.J.; Codée, J.D.C.; Overkleeft, H.S.; Van der Marel, G.A. Carbohydr. Res. 2007, 342, 419–429.

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Chapter 3    18 Fraser–Reid, B.; Wu, Z.F.; Andrews, C.W.; Skowronski, E.; Bowen, J.P. J. Am. Chem. Soc. 1991, 113, 1434–1435. 19 (a) Grice, P.; Ley, S.V.; Pietruszka, J.; Osborn, H.M.I.; Priepke, H.W.M.; Warriner, S.L. Chem. Eur. J. 1997, 3, 431–440 (b) Douglas, N.L.; Ley, S.V.; Lücking, U.; Warriner, S.L. J. Chem. Soc., Perkin Trans. 1 1998, 51–66 (c) Ley, S.V.; Baeschlin, D.K.; Dixon, D.J.; Foster, A.C.; Ince, S.J.; Priepke, H.W.M.; Reynolds, D.J. Chem. Rev. 2001, 101, 53–80 (d) Litjens, R.E.J.N.; Leeuwenburgh, M.A.; Van der Marel, G.A.; Van Boom, J.H. Tetrahedron Lett. 2001, 42, 8693–8696 (e) Crich, D.; De la Mora, M.; Vinod, A.U. J. Org. Chem. 2003, 68, 8142–8148 (f) Jensen, H.H.; Nordstrøm, L.U.; Bols, M. J. Am. Chem. Soc. 2004, 126, 9205–9213. 20 Dean, K.E.S.; Kirby, A.J.; Komarov, I.V. J. Chem. Soc., Perkin Trans. 2 2002, 337–341. 21 Bols and coworkers found that 1C4-locked methyl 3,6-anhydro-β-D-glucoside hydrolyses much faster than the corresponding non-locked 4C1 methyl β-D-glucoside: McDonnell, C.; López, O.; Murphy, P.; Fernández Bolaños, J.G.; Hazell, R.; Bols, M. J. Am. Chem. Soc. 2004, 126, 12374–12385. 22 (a) Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522 (b) Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. Tetrahedron 2003, 60, 1057–1064. 23 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326 24 Xu, L.; Price, N.P.J. Carbohydr. Res. 2004, 339, 1173–1178. 25 See Chapter 9 for mechanistic investigations. Comparing this coupling result with entry 3 in Table 2 (Chapter 2) revealed that this locked donor galactoside is more α-selective than the corresponding open form galacturonic acid ester. 26 Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819–6825. 27 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326. 28 Van den Bos, L.J.; Codée, J.D.C.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Biomol. Chem. 2003, 4160–4165. 29 (a) See Chapter 9 (b) Spijker, N.M.; Van Boeckel, C.A.A. Angew. Chem., Int. Ed. Engl. 1991, 30, 180–183.

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Chapter 4 │       

Stereocontrolled Synthesis of     β‐D‐Mannuronic Acid Esters:   Synthesis of an Alginate     Trisaccharide

Abstract: A facile route of synthesis towards β-linked mannuronic acid oligomers using the corresponding 1-thiomannuronic acid esters is presented. Activator systems used are the Ph2SO/Tf2O and NIS/TMSOTf reagent combinations. The presence of the remotely attached carboxylic ester sufficiently influences the electronic environment around the anomeric centre to allow good to excellent β-selectivities.1

Introduction Alginates are naturally occurring, linear polysaccharides composed of (1,4)-linked β-Dmannuronic acid and α-L-guluronic acid residues.2 They are produced by brown seaweeds of which three main polymer substructures have been reported, one containing mainly β-Dmannuronic acid residues, one containing mainly α-L-guluronic acid residues and a third composed of alternating sequences of β-D-mannuronic and α-L-guluronic acid. The Dmannuronic and L-guluronic residues are C5 epimers and possess different conformations: Dmannuronic acid adopts the 4C1 conformation and L-guluronic acid the 1C4 conformation. Due to their polyanionic character, alginates have found wide and diverse applications as thickening and gel-forming agents in food industry.3 Recently, it was discovered that mixtures of alginate oligomers have immunomodulating properties by binding to Toll-like receptors (TLRs) 2 and 4.4 TLRs are key players in mammalian immunity.5 They recognize molecules, most frequently from microbial origin,

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and instigate activation of inflammatory and antimicrobial innate immune responses. Additionally, recognition of microbial products by TLRs expressed on dendritic cells triggers functional maturation of dendritic cells and leads to initiation of antigen-specific adaptive immune responses. The availability of well-defined fragments of alginates and functionalized derivatives thereof would be highly useful in studying the mechanism of TLR-mediated recognition and ensuing signal transduction. To date, however, no synthesis of alginate oligomers has been reported. Both β-D-mannuronic and α-L-guluronic acid oligomers have a 1,2-cis relationship, which is difficult to attain in a stereocontrolled manner. The group of Crich has found an elegant solution for the introduction of the similarly challenging β-D-mannoside linkage by application of 2,3-di-O-benzyl-4,6-O-benzylidene protected 1-sulfoxide and 1-thiomannoside donors. 6,7 Upon activation of these donors, the 4,6-O-benzylidene moiety destabilizes the mannosyl oxacarbenium ion relative to the covalent α-mannosyl triflate intermediate.8 The latter is thought to undergo an SN2-type displacement with the incoming acceptor. The stereochemical influence of the benzylidene function was initially attributed to torsional effects, which prevents oxocarbenium ion formation at the anomeric center.6,9 Recently, however, Bols and coworkers showed that electronic effects are equally important for the observed high β-stereoselectivities.10,11 They found that the cyclic acetal locks the C5-C6 bond of the mannoside donor in the unfavorable tg conformation. The importance of electronic effects in β-mannoside synthesis is also highlighted by the application of compounds with non-participating, powerfully electron-withdrawing protective groups at the C2 position in combination with a good leaving group at C1 leading to good β-selectivities.12 In Chapter 2, an efficient route to suitably protected uronic acid donors by the application of 2,2,6,6-tetramethylpiperidinyloxy free radical (TEMPO)/[bis(acetoxy)iodo]benzene (BAIB) as a regio- and chemoselective oxidation system was disclosed.13 The presence of an electronwithdrawing carboxyl moiety at C5 makes uronic acid donors highly inreactive. A study towards the glycosylation properties of protected 1-thio glucuronic and galacturonic acid donors showed that these donors could be applied in sequential glycosylation strategies using sulfonium ion-based promoter systems.14 In this Chapter, it is demonstrated that 1-thio mannuronic acid esters are viable starting compounds for the construction of β-(1→4)-linked mannuronic acid oligomers.

Results and Discussion Thiomannuronic acid derivatives 6, 8 and 11 were selected as model compounds to investigate their glycosylation properties in terms of yield and stereoselectivity (Scheme 1). Known phenyl 1-thio-α-D-mannopyranoside 1 was converted by standard procedures into

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Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    15

16

benzylated derivatives 2 and 3. Compound 9 was readily obtained from phenyl 1-thio-α-Dgalactopyranoside (1) according to a procedure described by Kong and coworkers.17 Regioselective protection of the C6 and C3 positions as silyl-ethers was followed by benzylation of the residual C2 and C4 positions using BnBr and NaH. Removal of the TBSether functionalities gave the desired phenyl 2,4-di-O-benzyl-1-thio-α-D-mannopyranoside (9) in 30% yield. Contrary to what is reported by Kong and coworkers, also some 3,4-di-Obenzylated product was isolated. The next step was oxidation of substrates 2, 3 and 9 using the TEMPO/BAIB reagent system.13 In the case of compounds 2 and 3, the corresponding uronic acids were obtained (4 and 7, respectively), which were methylated using MeI/K2CO3 to give uronic ester donors 5 and 8. Acetylation of compound 5 using Ac2O/pyridine afforded donor 6. The 3,6-unprotected diol in compound 9 underwent tandem oxidation/lactonization affording lactone 10.14a,b Acidic opening of the lactone 10 using Amberlite H+/MeOH followed by acetylation yielded donor 11.

Scheme 1. O 4

O

SPh OB n

O

b from 9

1

R O 3 RO 2 RO

OR O

a from 1

4

OR O

1

10

e

2

3

4

1: R =R =R =R =H 1 3 2 4 9: R =R =B n, R =R =H

S Ph 1

2

3

4

2: R =R =Bn, R =R =H 1 2 3 4 3: R =R =R =Bn, R =H

c d

SPh 1 2 4: R =H, R =H 1 2 5: R =Me, R =H 1 2 6: R =Me, R =A c

from 3 b

f

MeO OC O Bn O BnO AcO 11

R OOC OBn 2 O R O BnO

b from 2

SPh

BnO

1

1

RO 3 R O 2 R O

SP h

RO OC O Bn O BnO Bn O

c

SP h 7: R=H 8: R=Me

Reagents and conditions: (a) for compound 2: see ref 15, for compound 3: see ref 16 (b) TEMPO, BAIB, DCM/H2O (2/1), 4: 87%, 7: 89%, 10: 63% (c) MeI, K2CO3, DMF, 5: 95%, 8: 93% (d) Ac2O, pyridine, quant. (e) 1) TBDMSCl, Imidazole, DMF, 2) BnBr, NaH, DMF, 3) TsOH, MeOH, reflux, 30% (over 3 steps) (f) 1) Amberlite IR120 H+, MeOH, 2) Ac2O, pyridine, 81% (2 steps).

With these donor building blocks in hand, the next objective was to determine their glycosylation properties in terms of yield and stereoselectivity (Table 1). Pre-activation of 4O-acetyl donor 6 using diphenylsulfonium bistriflate, generated in situ at -50°C, and subsequent slow addition of acceptor 12 afforded β-linked disaccharide 13 (1JC,H = 156 Hz)18 in 81% yield. Condensation of the 3-O-acetyl donor 11 with acceptor 12 led to α-dimer 14 (1JC,H = 175 Hz), whereas condensation of tri-O-benzyl donor 8 with the sterically more demanding acceptor 15 furnished β-dimer 16 (1JC,H = 159 Hz).19 The stereochemical outcome of these condensations indicate that the 3-O-acetyl moiety may participate through a

67   

67

Chapter 4    Table 1.

entry

1

donor MeOOC OBn O AcO BnO 6

2

acceptor

activator yield (α/β)a,b

OH

Ph2SO/Tf2O 81% (0/1)

BnO

SPh

12

MeOOC OBn O BnO AcO 11

O

BnO BnO

OMe

disaccharide MeOOC OBn O AcO BnO

NIS/TMSOTf 91% (0/1)

13

MeOOC OBn O BnO AcO

12

MeOOC OBn O BnO BnO 8

O OBn BnO

Ph

O

O O

OpMP

HO

Ph2SO/Tf2O 55% (0/1)

OBn

SPh

MeOOC OBn O BnO BnO

15

6

O

O O

15

NIS/TMSOTf 58% (0/1)

OpMP

O OBn

16

Ph

Ph2SO/Tf2O 69% (0/1) 4

OMe OBn

O

14

Ph

3

OMe OBn

OBn

BnO

Ph2SO/Tf2O 66% (1/0)

SPh

O

O

MeOOC OBn O AcO BnO

O O O

OpMP

O OBn

17

O O

5

6

H

O

O

HO 18

HO

6

a

6

COOMe O

BnO OBn 20

O

Ph2SO/Tf2O 71% (0/1)

MeOOC OBn O AcO BnO

O

O

H

19

O

O O O

Ph2SO/Tf2O no rxn

MeOOC OBn O AcO BnO O

OpMP

NIS/TMSOTf 99% (1/2)

COOMe O

BnO 21

OpMP

OBn

isolated yields. b anomeric ratios were determined by 1H NMR spectroscopy.

6-membered transition state, thereby preventing β-side attack.20 The glycosylation properties of 4-O-acetyl donor 6 were further evaluated by executing condensation reactions with the less reactive and therefore more α-directing glycosyl acceptors 15, 18 and 20.21 The condensations of 6 with 15 and 18 led to the isolation of β-disaccharides 17 (69%) and 19 (71%), respectively. Subjection of the sterically and electronically deactivated acceptor 20 to 68   

68

Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters   

the glycosylation protocol did not lead to a productive coupling, indicating that the reactivity of the acceptor plays a decisive role on the outcome of a glycosylation reaction.12c The finding that the introduction of 1,2-cis-equatorial glycosidic linkages can be accomplished with different types of donors and promotor systems led us to explore NIS/TMSOTf-mediated glycosylation reactions.22,23 In a first attempt, a mixture of donor 6, acceptor 12 and NIS in DCM was cooled to -40°C and a catalytic amount of TMSOTf was added affording β-disaccharide 13 in 91% yield. In another experiment, donor 6 was preactivated with NIS and an equimolar amount of TMSOTf.23 However, no complete activation of donor 6 could be achieved as monitored by TLC. After addition of acceptor 12, the βlinked disaccharide 13 was isolated in the same yield (91%). These results suggest that, contrary to the Ph2SO/Tf2O conditions, the donor is not transformed into a α-triflate intermediate. Possibly, direct SN2-type displacement of the initially formed iodosulfonium species is at the basis of the observed β-selectivities in the NIS/TMSOTf-mediated glycosylation reactions. The standard NIS/TMSOTf protocol was also applied for the condensation of donor 6 and benzylidene-protected acceptor 15 to give β-linked disaccharide 16 in 58% yield. Partial cleavage of the acid labile benzylidene functionality, observed by TLC-analysis, may explain this moderate yield. Finally, the earlier unproductive coupling of acceptor 20 with donor 6 was executed using NIS/TMSOTf. Disaccharide 21 was isolated in excellent yield as an anomeric mixture (α/β = 1/2), probably as a result of the sterical bias in the acceptor.

Scheme 2. MeOOC OBn O LevO BnO

a 5

22

MeOOC OBn O RO BnO

d

MeOOC OBn O R2O BnO

b 6

MeOOC OBn O O BnO

1

R OOC OR O R2O R1O

g

R3OOC OR1 O O R1O

O N3

25: R=Lev 26: R=H

e

OR1 23: R1=α/β-O(CH2)3N3, R2=Ac 24: R1=β-O(CH2)3N3, R2=H

c

3

SPh

MeOOC OBn O LevO BnO R3OOC OR1 O O R1O

f

22

SPh

O

27: R1=Bn, R2=Lev, R3=Me, R4=N3 28: R1=R2=R3=H, R4=NH2

R4

Reagents and conditions: (a) Lev2O, pyridine, 86% (b) 6, Ph2SO, Tf2O, TTBP, DCM, -75°C then 3azidopropanol, (α/β = 1/5), 80% (c) NaOMe, MeOH, 60% (d) 22, 24, NIS, TMSOTf, DCM, -40°C, 78% (α/β = 0/1) (e) NH2NH2·H2O, pyridine, AcOH, 95% (f) 22, 26, NIS, TMSOTf, DCM, -40°C, 50% (α/β = 1/>10) (g) 1) NH2NH2·H2O, pyridine, AcOH, 2) KOH, H2O, THF, 3) H2, Pd/C (10%), MeOH/AcOH (10/1), 35% (over 3 steps).

69   

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Chapter 4   

The stage was now set for the assembly of the spacer containing mannuronic acid trisaccharide 28 using the NIS/TMSOTf activation system (Scheme 2). Levulinoylation of compound 5 using Lev2O in pyridine afforded donor 22 in excellent yield. The requisite acceptor 24 was synthesized under Ph2SO/Tf2O-mediated conditions from donor 6 with 3azidopropanol affording fully protected dimer 23 in 80% yield as an inseparable mixture of anomeric (α/β = 1/5).24 Ensuing basic hydrolysis of the acetate ester using NaOMe in MeOH delivered the target β-linked acceptor 24 in 60% yield. It was found that hydrolysis of the βanomer 23β occurred much faster than hydrolysis of the corresponding α-oriented anomer 23α, resulting in facile separation of the anomers. Donor 22 and acceptor 24 were subjected to the NIS/TMSOTf-mediated condensation giving β-linked disaccharide 25 in 78% yield. Standard deprotection of the levulinoyl group using hydrazine hydrate yielded acceptor disaccharide 26 which was subjected to the same coupling cycle to give trisaccharide 27 in 50% yield. Global deprotection of the mannuronic acid trisaccharide 27 was accomplished via levulinoyl cleavage, hydrolysis of the methyl ester and final reduction of the azide and benzyl groups. HW40 gel filtration afforded deprotected trisaccharide 28 in 35% yield over the three steps.

Conclusion This Chapter describes a facile synthesis route towards β-1,4-mannuronic acid containing diand trimers. The carboxylic ester function in the mannuronic acid donors sufficiently influences the electronic environment around the anomeric centre to allow good to excellent β-selectivities in Ph2SO/Tf2O and NIS/TMSOTf-mediated glycosylation events. This strategy is a valuable asset for future assembly of alginate oligomers, which may facilitate further studies into their properties as ligands for Toll-like receptors 2 and 4.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Brüker DMX-400 and a Brüker AV-400 (400/100 MHz), Brüker AV500 (500/125 MHz) and a Brüker DMX-600 (600/150 MHz) spectrometer. Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and Q-Star Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide and TTBP were removed by co-evaporation with toluene. TTBP was synthesized as described by Crich et al.25 Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography were of pro analysi

70   

70

Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    quality. Flash chromatography was performed on Fluka silica gel 60 (0.04 – 0.063 mm). TLC-analysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) were applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in water followed by charring at ~150°C. All reactions were performed under an inert atmosphere of Argon unless stated otherwise. N.B.: NMR-spectra of the various uronates showed low intensity of the conformational equilibria.

13

C signals, probably due to

General Procedure for the TEMPO/BAIB-Mediated Oxidation: To a vigorously stirred solution of 0.3 mmol thioglycoside in 1 mL DCM and 0.5 mL H2O was added 0.06 mmol TEMPO (0.2 equiv.) and 0.75 mmol BAIB (2.5 equiv.). Stirring was allowed until TLC indicated complete conversion of the starting material to a lower running spot (~45min). The reaction mixture was quenched by the addition of 10 mL Na2S2O3 solution (10% in H2O) and 10 mL NaHCO3 (sat. aq.). The mixture was then extracted twice with EtOAc (10 mL) and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash column chromatography using EtOAc/petroleum ether (for the uronic acids 2% (v/v) AcOH is used) afforded the pure oxidized products. General Procedure for Glycosidations using Ph2SO/Tf2O: A solution of 1-thio mannopyranoside uronic acid ester (1 equiv.), diphenyl sulfoxide (1.3 equiv.) and tri-tert-butylpyrimidine (2.5 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30min. The mixture was cooled to -60°C before triflic acid anhydride (1.3 equiv.) was added. The mixture was allowed to warm to -45°C in 15min followed by addition of acceptor (1.5 equiv.) in DCM (0.15M). Stirring was continued and the reaction mixture was allowed to warm to rT. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding disaccharides. General Procedure for Glycosidations using NIS/TMSOTf: Method A (pre-mixing donor and acceptor): A solution of 1-thio mannopyranoside uronic acid ester (1 equiv.) and acceptor (1.5 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30min before Niodosuccinimide (1.3 equiv.) was added. The mixture was cooled to -40°C followed by the addition of trimethylsilyl trifluoromethanesulfonate (0.1 equiv.). Stirring was continued and the reaction mixture was allowed to warm to rT. When TLC-analysis indicated complete reaction, triethylamine (5 equiv.) was added. The reaction mixture was diluted with EtOAc and washed with Na2S2O3 (10% in H2O). The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding disaccharides. Method B (pre-activation using NIS and equimolar TMSOTf): A solution of 1-thio mannopyranoside uronic acid ester (1 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30min before N-iodosuccinimide (1.3 equiv.) was added. The mixture was cooled to -40°C before trimethylsilyl trifluoromethanesulfonate (1.3 equiv.) was added. The mixture was allowed to warm to -30°C in 15min followed by addition of acceptor (1.5 equiv.) in DCM (0.15M). Stirring was continued and the reaction mixture was allowed to warm to rT. When TLC-analysis indicated complete reaction triethylamine (5 equiv.) was added. The reaction mixture was diluted with EtOAc

71   

71

Chapter 4    and washed with Na2S2O3 (10% in H2O). The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding disaccharides.

Phenyl 2,3-di-O-benzyl-1-thio-α-D-mannopyranosiduronic acid (4): Diol 215 was converted according to the general procedure delivering 1-thio mannuronic acid 4 in 87% yield as a SPh colorless oil. TLC: 50% EtOAc/PE (5% AcOH); [α]D22: +16° (c = 0.4, CHCl3); 1H NMR (500 MHz, MeOD) δ = 3.72 (dd, 1H, J = 8.0 Hz, J = 3.0 Hz, H-3), 3.94 (t, 1H, J = 3.5 Hz, H-2), 4.26 (dd, 1H, J = 8.0 HOOC OBn O HO BnO

Hz, J = 7.5 Hz, H-4), 4.43 (d, 1H, J = 7.0 Hz, H-5), 4.56 (d, 1H, J = 10.5 Hz, CHHPh), 4.60 (d, 1H, J = 10.5 Hz, CHHPh), 4.63 (d, 1H, J = 10.5 Hz, CHHPh), 4.71 (d, 1H, J = 10.5 Hz, CHHPh), 5.61 (d, 1H, J = 3.5 Hz, H-1), 7.27 – 7.59 (m, 15H, H Arom); 13C NMR (100 MHz, MeOD) δ = 69.3 (C-4), 73.2 (CH2 Bn), 73.5 (CH2 Bn), 75.5 (C-5), 76.8 (C-2), 79.0 (C-3), 86.2 (C-1), 128.4 – 135.2 (CH Arom), 139.3 (Cq Bn), 139.5 (Cq Bn), 172.5 (C=O COOMe); 13C-GATED (125 MHz, MeOD): 86.2 (J = 167 Hz, C-1); HRMS: [M+Na]+ calcd for C26H26O6SNa 489.13423, found 489.13446. MeOOC OBn O HO BnO SPh

Methyl (phenyl 2,3-di-O-benzyl-1-thio-α-D-mannopyranoside) uronate (5): To a stirred solution of 965 mg compound 4 (2.1 mmol) in 10 mL DMF was added 0.3 mL MeI (5 mmol, 2.5 equiv.) and 200 mg K2CO3 (excess). The solution was stirred overnight and quenched by

the addition of 2 mL MeOH. The reaction mixture was taken up in EtOAc and washed with H2O. The aqueous phase was extracted with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/PE) afforded 0.96 g of the title compound 5 (1.9 mmol, 95%) as a colorless oil. TLC: 35% EtOAc/PE; [α]D22: +40° (c = 1, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.98 (s, 1H, OH-4), 3.71 (dd, 1H, J = 7.2 Hz, J = 2.4 Hz, H-3), 3.78 (s, 3H, CH3 COOMe), 3.95 (t, 1H, J = 2.4 Hz, J = 1.6 Hz, H-2), 4.38 (t, 1H, J = 7.2 Hz, H-4), 4.57 (d, 1H, J = 9.6 Hz, CHHPh), 4.60 (d, 1H, J = 9.6 Hz, CHHPh), 4.61 (s, 1H, H-5), 4.66 (d, 1H, J = 9.6 Hz, CHHPh), 4.69 (d, 1H, J = 9.6 Hz, CHHPh), 5.61 (d, 1H, J = 1.6 Hz, H-1), 7.24 – 7.45 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.6 (CH3 COOMe), 68.5 (C-4), 72.3 (CH2 Bn), 72.5 (CH2 Bn), 72.6 (C-5), 75.7 (C-2), 78.1 (C-3), 86.2 (C-1), 127.6 – 131.5 (CH Arom), 133.7 (Cq SPh), 137.6 (Cq Bn), 137.9 (Cq Bn), 170.3 (C=O COOMe); HRMS: [M+Na]+ calcd for C27H28O6SNa 503.14988, found 503.14992. MeOOC OBn O AcO BnO

Methyl (phenyl 4-O-acetyl-2,3-di-O-benzyl-1-thio-α-D-mannopyranoside) uronate (6): Compound 5 (0.96 g, 1.9 mmol) was treated with 3 mL pyridine/Ac2O (3:1) solution for 8h

SPh followed by the addition 2 mL MeOH. The reaction mixture was taken up in EtOAc and washed with 2M HCl and saturated aq. NaHCO3. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/PE) afforded 1.04 g of the title compound 6 (1.9 mmol, quant.) as a colorless oil. TLC: 25% EtOAc/PE; [α]D22: +28° (c = 1, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.03 (s, 3H, CH3 Ac), 3.60 (s, 3H, CH3 COOMe), 3.75 (d, 1H, J = 5.0 Hz, H-2), 3.80 (dd,

1H, J = 6.0 Hz, J = 2.8 Hz, H-3), 4.49 (d, 1H, J = 11.9 Hz, CHHPh), 4.54 (d, 1H, J = 4.5 Hz, H-5), 4.57 (d, 1H, J = 12.1 Hz, CHHPh), 4.61 (d, 1H, J = 12.1 Hz, CHHPh), 4.63 (d, 1H, J = 11.9 Hz, CHHPh), 5.55 (t, 1H, J = 6.0 Hz, J = 4.5 Hz, H-4), 5.77 (d, 1H, J = 7.0 Hz, H-1), 7.22 – 7.59 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 52.4 (CH3 COOMe), 69.4 (C-4), 72.4 (CH2 Bn), 72.7 (C-5), 73.1 (C-2, C-3), 82.9 (C-1), 126.7 – 131.3 (CH Arom), 133.6 (Cq SPh), 137.3 (Cq Bn), 137.4 (Cq Bn), 168.4 (C=O Ac or COOMe), 169.6 (C=O COOMe or Ac); 13C-GATED (100 MHz, CDCl3): 82.9 (J = 168 Hz, C-1); HRMS: [M+NH4]+ calcd for C29H34O7SN 540.20560, found 540.20614.

72   

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Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    16

Phenyl 2,3,4-tri-O-benzyl-1-thio-α-D-mannopyranosiduronic acid (7): Compound 3 was converted according to the general procedure delivering title compound 7 in 68% yield as a SPh colorless oil. TLC: 25% EtOAc/PE; [α]D22: +34° (c = 1, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 3.81 (dd, 1H, J = 6.8 Hz, J = 2.8 Hz, H-3), 3.91 (dd, 1H, J = 5.2 Hz, J = 2.8 Hz, H-2), 4.23 (t, 1H, J = 6.8 HOOC OBn O BnO BnO

Hz, H-4), 4.52 (d, 1H, J = 12.0 Hz, CHHPh), 4.54 (d, 1H, J = 12.0 Hz, CHHPh), 4.57 (d, 1H, J = 12.0 Hz, CHHPh), 4.64 (d, 1H, J = 6.8 Hz, H-5), 4.65 – 4.73 (m, 3H, 2xCHHPh, CHHPh), 5.67 (d, 1H, J = 5.2 Hz, H-1), 7.22 – 7.51 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3)26 δ = 72.0, 72.3 (CH2 Bn), 72.3 (CH2 Bn), 74.9, 75.7 127.4 – 131.5 (CH Arom), 133.5 (Cq SPh), 137.5 (Cq Bn), 137.7 (Cq Bn), 172.4 (C=O COOH); HRMS: [M+Na]+ calcd for C33H32O6SNa 579.18118 found 579.18222. Methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-α-D-mannopyranoside) uronate (8): To a stirred solution of 373 mg compound 7 (0.67 mmol) in 2.2 mL DMF was added 92 µL MeI (1.5 SPh mmol, 2 equiv.) and 200 mg K2CO3 (excess). The solution was stirred overnight and quenched by the addition of 2 mL MeOH. The reaction mixture was diluted with EtOAc and washed with H2O. The MeOOC OBn O BnO BnO

aqueous phase was extracted with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/PE) afforded 355 mg of the title compound 8 (0.62 mmol, 92%) as a colorless oil. TLC: 25% EtOAc/PE; 1H NMR (400 MHz, CDCl3) δ = 3.46 (s, 3H, CH3 COOMe), 3.81 (dd, 1H, J = 7.0 Hz, J = 2.8 Hz, H-3), 3.89 (dd, 1H, J = 5.2 Hz, J = 2.8 Hz, H-2), 4.26 (t, 1H, J = 7.0 Hz, J = 6.2 Hz, H-4), 4.52 (d, 1H, J = 12.0 Hz, CHHPh), 4.55 (s, 2H, CH2 Bn), 4.63 (d, 1H, J = 6.2 Hz, H-5), 4.64 – 4.69 (m, 3H, 2xCHHPh, CHHPh), 5.67 (d, 1H, J = 5.2 Hz, H-1), 7.23 – 7.53 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.2 (CH3 COOMe), 72.1 (CH2 Bn), 72.4 (CH2 Bn), 72.9 (C-5), 73.7 (CH2 Bn), 74.9 (C-2), 75.6 (C-4), 77.1 (C-3), 84.7 (C-1), 127.2 – 131.4 (CH Arom), 134.9 (Cq SPh), 137.8 (2xCq Bn), 137.9 (Cq Bn), 169.2 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 84.7 (J = 163 Hz, C-1); HRMS: [M+Na]+ calcd for C34H34O6SNa 593.19683 found 593.19780. HO BnO HO

OBn O SPh

Phenyl 2,4-di-O-benzyl-α-D-mannopyranoside (9): To a cooled solution (0°C) of 2.71 g compound 1 (10.0 mmol) in 50 mL DMF was added 3.24 g TBSCl (21.5 mmol, 2.15 equiv.) and 3.18 g imidazole (21.5 mmol, 2.15 equiv.). The mixture was allowed to warm to room

temperature. After stirring for 8 h the reaction was quenched with MeOH, taken up in EtOAc and washed with H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude product was filtered through a plug of silica gel using (10% EtOAc/PE) as the eluent. After evaporation the product was dissolved in 50 mL DMF and cooled to 0°C before 3 mL BnBr (25 mmol, 2.5 equiv.) and 1 g NaH (60% in min. oil, 25 mmol, 2.5 equiv.) were added. The mixture was allowed to stir for 12h and the reaction was quenched by MeOH, taken up in Et2O and washed with H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The oily substance was suspended in 50 mL MeOH and a catalytic amount of TsOH was added. The mixture was refluxed until TLC indicated complete conversion. After addition of 5 mL NEt3 the reaction mixture was concentrated under reduced pressure. Flash chromatography afforded 1.35 g of the title compound 9 (2.98 mmol, 30%) as a colorless oil. TLC: 50% EtOAc/PE; [α]D22: +59° (c = 1, CHCl3); 1H NMR (600 MHz, CDCl3) δ = 1.84 (bs, 1H, OH-6), 2.42 (d, 1H, J = 7.6 Hz, OH-3), 3.74 (t, 1H, J = 9.6 Hz, H-4), 3.78 – 3.85 (m, 2H, H-6), 4.00 (bs, 2H, H-2, H-3), 4.12 (dt, 1H, J = 4.0 Hz, J = 3.2 Hz, H-5), 4.56 (d, 1H, J = 11.6 Hz, CHHPh), 4.67 (d, 1H, J = 11.6 Hz, CHHPh), 4.75 (d, 1H, J = 11.6 Hz, CHHPh), 4.93 (d, 1H, J = 11.6 Hz, CHHPh), 5.57 (s, 1H, H-1), 7.24 – 7.48 (m, 15H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 62.1 (C-6), 72.2 (C-2 or C-3), 72.4 (C-5), 72.5 (CH2 Bn), 74.9 (CH2 Bn), 76.4 (C-4), 79.7 (C-3 or C-2), 85.1 (C-1), 127.4 – 131.9 (CH Arom), 133.6 (Cq SPh), 137.2 (Cq Bn), 138.1 (Cq Bn); 13C-GATED (150 MHz, CDCl3): 85.1 (J = 165 Hz, C-1); HRMS: [M+Na]+ calcd for C26H28O5SNa 475.15497, found 475.15565.

73   

73

Chapter 4    O O O

SPh OBn

BnO

Phenyl 2,4-di-O-benzyl-1-thio-α-D-mannopyranosidurono-6,3-lactone (10): Diol 9 was converted according to the general procedure delivering 1-thio mannuronic acid lactone 10 in 63% yield as a colorless oil. TLC: 25% EtOAc/PE; 1H NMR (500 MHz, CDCl3) δ = 3.94 (dd, 1H, J = 9.0 Hz, J = 1.0 Hz, H-2), 4.03 (dd, 1H, J = 6.0 Hz, J = 3.0 Hz, H-4), 4.35 (d, 1H, J =

2.5 Hz, H-5), 4.44 (d, 1H, J = 11.5 Hz, CHHPh), 4.58 (d, 1H, J = 11.5 Hz, CHHPh), 4.66 (d, 1H, J = 11.5 Hz, CHHPh), 4.70 (d, 1H, J = 6.0 Hz, H-3), 4.78 (d, 1H, J = 11.5 Hz, CHHPh), 5.00 (d, 1H, J = 9.0 Hz, H-1), 7.24 – 7.65 (m, 15H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 71.7 (CH2 Bn), 72.0 (C-5), 72.9 (CH2 Bn), 73.1 (C-2), 74.9 (C-4), 77.8 (C-3), 84.3 (C-1), 127.8 – 132.4 (CH Arom), 132.1 (Cq SPh), 136.3 (Cq Bn), 137.2 (Cq Bn), 169.6 (C=O lactone); 13C-GATED (125 MHz, CDCl3): 84.3 (J = 158 Hz, C-1); HRMS: [M+H]+ calcd for C26H25O5S 448.13444, found 448.19671. MeOOC OBn O BnO AcO SPh

Methyl (phenyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-α-D-mannopyranoside) uronate (11): Compound 10 (170 mg, 0.37 mmol) was dissolved in MeOH and, after addition of 200 mg Amberlite IR120 H+ resin, heated under reflux. After 4h, TLC analysis indicated complete

conversion of starting material into a lower running product. The reaction mixture was filtered and concentrated under reduced pressure. The product was treated with 5 mL pyridine/Ac2O (3:1) for 8h followed by addition of MeOH. The reaction mixture was diluted with EtOAc and washed with 2M HCl and sat. NaHCO3. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/PE) afforded 156 mg of the title compound 11 (0.3 mmol, 81%) as a colorless oil. TLC: 25% EtOAc/PE; [α]D22: +29° (c = 1.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 1.97 (s, 3H, CH3 Ac), 3.72 (CH3 COOMe), 3.99 (dd, 1H, J = 5.1 Hz, J = 3.1 Hz, H-2), 4.25 (t, 1H, J = 6.9 Hz, H-4), 4.48 (d, 1H, J = 11.8 Hz, CHHPh), 4.59 (d, 1H, J = 11.8 Hz, CHHPh), 4.61 (d, 1H, J = 11.4 Hz, CHHPh), 4.67 (d, 1H, J = 11.4 Hz, CHHPh), 4.68 (d, 1H, J = 6.9 Hz, H-5), 5.25 (dd, 1H, J = 7.3 Hz, J = 3.1 Hz, H-3), 5.58 (d, 1H, J = 5.1 Hz, H-1), 7.24 – 7.56 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 52.4 (CH3 COOMe), 70.9 (C3), 72.3 (CH2 Bn), 72.7 (C-5), 73.8 (CH2 Bn), 74.6 (C-2), 75.0 (C-4), 84.1 (C-1), 127.5 – 131.7 (CH Arom), 133.3 (Cq SPh), 137.3 (Cq Bn), 137.6 (Cq Bn), 169.4 (C=O Ac or COOMe), 169.7 (C=O COOMe or Ac); 13CGATED (100 MHz, CDCl3): 84.1 (J = 165 Hz, C-1); HRMS: [M+NH4]+ calcd for C29H34O7SN 540.20560, found 540.20607. MeOOC OBn O AcO BnO

O

O

BnO

OBn

OMe OBn

Methyl (2,3,4-tri-O-benzyl-6-O-(methyl 4-O-acetyl-2,3-di-O-benzyl-β-D-mannopyranosyluronate)-α-D-glucopyranoside (13): Donor 6 was glycosylated with acceptor 12 in the same way as described in the general procedure (Ph2SO/Tf2O) delivering the title compound 13 as a colorless oil. TLC: 30% EtOAc/PE; [α]D22:

-11° (c = 0.6, CHCl3); 1H NMR (500 MHz, CDCl3) δ = 2.02 (s, 3H, CH3 Ac), 3.31 (s, 3H, CH3 OMe), 3.37 (dd, 1H, J = 9.5 Hz, J = 2.5 Hz, H-3’), 3.41 (m, 2H, H-4 and H-6), 3.50 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-2), 3.71 (s, 4H, H-2’ and CH3 COOMe), 3.75 (s, 1H, H-5’), 3.79 (bt, 1H, J = 8.0 Hz, J = 6.0 Hz, H-5), 4.01 (t, 1H, J = 9.5 Hz, H-3), 4.12 (bs, 2H, H-1’ and H-6), 4.36 (d, 1H, J = 12.5 Hz, CHHPh), 4.47 (d, 1H, J = 12.5 Hz, CHHPh), 4.49 (d, 1H, J = 12.5 Hz, CHHPh), 4.56 (d, 1H, J = 3.5 Hz, H-1), 4.66 (d, 1H, J = 12.0 Hz, CHHPh), 4.75 (d, 1H, J = 11.5 Hz, CHHPh), 4.79 (d, 1H, J = 12.0 Hz, CHHPh), 4.81 (d, 1H, J = 11.5 Hz, CHHPh), 4.84 (d, 1H, J = 10.5 Hz, CHHPh), 4.90 (d, 1H. J = 12.5 Hz, CHHPh), 5.01 (d, 1H, J = 10.5 Hz, CHHPh), 5.48 (t, 1H, J = 9.5 Hz, H-4’), 7.18 – 7.37 (m, 25H H Arom); 13C NMR (125 MHz, CDCl3) δ = 20.8 (CH3 Ac), 48.5 (CH3 COOMe), 52.5 (CH3 OMe), 68.8 (C-6), 68.9 (C-4’), 69.7 (C-5), 71.5 (CH2 Bn), 72.9 (C-2’), 73.3 (CH2 Bn), 73.6 (CH2 Bn), 73.7 (C-5’), 74.7 (CH2 Bn), 75.7 (CH2 Bn), 77.6 (C-4), 78.2 (C-3’), 79.8 (C-2), 82.1 (C-3), 97.8 (C-1), 101.6 (C1’), 127.4 – 128.5 (CH Arom), 137.6 (Cq Bn), 138.0 (Cq Bn), 138.3 (2xCq Bn), 138.7 (Cq Bn), 167.9 (C=O Ac or

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Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    13

COOMe), 169.5 (C=O Ac or COOMe); C-GATED (125 MHz, CDCl3): 97.8 (JC1,H1 = 169 Hz, C-1), 101.6 (JC1’,H1’ = 156 Hz, C-1’); HRMS: [M+NH4]+ calcd for C51H60O13N 894.40647, found 894.40531. N.B.: Conclusive evidence for the β-orientation was gained by reducing both ester functions after which the heteronuclear coupling constant was determined at 1JC1’,H1’ = 153 Hz. MeOOC OBn O BnO AcO O

O

OMe OBn

Methyl (2,3,4-tri-O-benzyl-6-O-(methyl 4-O-acetyl-2,3-di-O-benzyl-α-D-mannopyranosyluronate)-α-D-glucopyranoside (14): Donor 11 was glycosylated with acceptor 12 in the same way as described in the general procedure (Ph2SO/Tf2O)

delivering the title compound 14 as a colorless oil. TLC: 30% EtOAc/PE; 1H NMR BnO (400 MHz, CDCl3) δ = 1.96 (s, 3H, CH3 Ac), 3.36 (s, 3H, CH3 OMe), 3.48 (t, 1H, J = 9.2 Hz, H-4), 3.50 (dd, 1H, J = 9.6 Hz, J = 3.6 Hz, H-2), 3.68 (s, 3H, CH3 COOMe), 3.71 – 3.77 (m, 2H, H-5, H-6), 3.86 (t, 1H, J = 3.0 Hz, H-2’), 3.90 (dd, 1H, J = 11.1 Hz, J = 4.8 Hz, H-6), 3.99 (t, 1H, J = 9.6 Hz, J = 9.2 Hz, H-3), 4.19 (t, 1H, J = 8.7 Hz, H-4’), 4.29 (d, 1H, J = 8.7 Hz, H-5’), 4.53 (d, 1H, J = 12.3 Hz, CHHPh), 4.56 OBn

(d, 1H, J = 3.6 Hz, H-1), 4.61 (d, 1H, J = 10.9 Hz, CHHPh), 4.63 (d, 1H, J = 12.0 Hz, CHHPh), 4.65 (s, 2H, CH2 Bn), 4.67 (d, 1H, J = 12.3 CHHPh), 4.78 (d, 1H, J = 12.0 Hz, CHHPh), 4.82 (d, 1H, J = 10.9 Hz, CHHPh), 4.92 (d, 1H, J = 10.9 Hz, CHHPh), 4.99 (d, 1H, J = 11.1 Hz, CHHPh), 5.00 (s, 1H, H-1’), 5.24 (dd, 1H, J = 8.6 Hz, J = 3.2 Hz, H-3’), 7.23 – 7.38 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.9 (CH3 Ac), 52.3 (CH3 OMe), 55.1 (CH3 COOMe), 66.7 (C-6), 69.8 (C-5), 71.5 (C-5’), 72.5 (C-3’), 72.9 (CH2 Bn), 73.3 (CH2 Bn), 74.3 (CH2 Bn), 74.8 (C-4’), 75.0 (CH2 Bn), 75.0 (C-2’), 75.7 (CH2 Bn), 77.6 (C-4), 79.9 (C-2), 82.1 (C-3), 97.8 (C-1), 98.5 (C-1’); 127.5 – 129.1 (CH Arom), 137.7 (Cq Bn), 137.9 (Cq Bn), 138.1 (Cq Bn), 138.6 (Cq Bn), 169.6 (C=O COOMe or Ac), 169.9 (C=O Ac or COOMe); 13C-GATED (100 MHz, CDCl3): 97.8 (JC1,H1 = 166 Hz, C-1), 98.5 (JC1’,H1’ = 170 Hz, C-1’); HRMS: [M+NH4]+ calcd for C51H60O13N 894.40647, found 894.40545.

Ph

MeOOC OBn O BnO BnO

O

O O

O

OpMP

para-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-3-O-(methyl 2,3,4-tri-Obenzyl-β-D-mannopyranosyluronate)-β-D-galactopyranoside (16): Donor 8 was glycosylated with acceptor 15 in the same way as described in the general procedure (Ph2SO/Tf2O) delivering the title compound 16 as a colorless oil.

OBn

TLC: 30% EtOAc/PE; [α]D22: -4° (c = 0.5, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 3.16 (dd, 1H, J = 9.6 Hz, J = 2.9 Hz, H-3’), 3.56 (s, 1H, H-5), 3.63 (d, 1H, J = 2.8 Hz, H-2’), 3.72 (s, 3H, CH3 COOMe or OMe), 3.73 (d, 1H, J = 9.2 Hz, H-5’), 3.79 (s, 3H, CH3 COOMe or OMe), 3.84 (dd, 1H, J = 9.9 Hz, J = 3.4 Hz, H-3), 4.08 (dd, 1H, J = 9.9 Hz, J = 7.6 Hz, H-2), 4.09 (dd, 1H, J = 12.3 Hz, J = 1.7 Hz, H-6), 4.18 (t, 1H, J = 9.7 Hz, H-4’), 4.28 (d, 1H, J = 11.6 Hz, CHHPh), 4.30 (d, 1H, J = 11.3 Hz, CHHPh), 4.36 (d, 1H, J = 12.3 Hz, H-6), 4.37 (d, 1H, J = 11.6 Hz, CHHPh), 4.39 (d, 1H, J = 3.4 Hz, H-4), 4.61 (d, 1H, J = 10.7 Hz, CHHPh), 4.71 (s, 1H, H-1’), 4.85 (d, 1H, J = 10.7 CHHPh), 4.91 (d, 1H, J = 7.6 Hz, H-1), 4.93 (s, 2H, CH2 Bn), 4.96 (d, 1H, J = 11.6 Hz, CHHPh), 5.62 (s, 1H, CHPh), 6.84 (d, 2H, J = 9.0 Hz, H Arom pMP), 7.05 (d, 2H, J = 9.0 Hz, H Arom pMP), 7.10 – 7.60 (m, 25H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.3 (CH3 OMe), 55.6 (CH3 COOMe), 66.7 (C-5), 68.9 (C-6), 71.8 (CH2 Bn), 71.9 (C-2’), 73.4 (CH2 Bn), 75.2 (2xCH2 Bn), 75.3 (C-5’), 75.7 (C-4’), 75.8 (C-4), 77.7 (C-3), 79.0 (C-2), 82.0 (C-3’), 100.8 (CHPh), 103.2 (C-1 and C-1’), 114.5 (CH Arom pMP), 118.9 (CH Arom pMP), 126.3 – 128.7 (CH Arom), 137.9 (Cq Bn), 138.1 (Cq Bn), 138.3 (Cq Bn), 138.4 (Cq Bn), 151.5 (Cq pMP), 155.4 (Cq pMP), 168.7 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 103.2 (JC1’,H1’ = 159 Hz, C-1’), 103.2 (JC1,H1 = 159 Hz, C-1); HRMS: [M+Na]+ calcd for C55H56O13Na 947.36131, found 947.36288.

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Chapter 4    Ph O O

MeOOC OBn O AcO BnO

O

OpMP

O OBn

para-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-3-O-(methyl 4-O-acetyl2,3-di-O-benzyl-β-D-mannopyranosyluronate)-β-D-galactopyranoside (17): Donor 6 was glycosylated with acceptor 15 in the same way as described in the general procedure (Ph2SO/Tf2O and NIS/TMSOTf) delivering the title

compound 17 as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: -28° (c = 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 1.97 (s, 3H, CH3 Ac), 3.12 (dd, 1H, J = 9.8 Hz, J = 2.8 Hz, H-3’), 3.53 (s, 1H, H-5), 3.65 (d, 1H, J = 2.7 Hz, H-2’), 3.70 (d, 1H, J = 9.7 Hz, H-5’), 3.72 (s, 3H, CH3 COOMe or OMe), 3.78 (s, 3H, CH3 OMe or COOMe), 3.83 (dd, 1H, J = 9.9 Hz, J = 3.4 Hz, H-3), 4.07 (t, 1H, J = 9.9 Hz, H-2), 4.10 (m, 1H, H-6), 4.18 (d, 1H, J = 12.1 Hz, CHHPh), 4.30 (d, 1H, J = 12.4 Hz, CHHPh), 4.37 (m, 3H, H-6, 2xCHHPh), 4.42 (d, 1H, J = 3.4 Hz, H-4), 4.70 (s, 1H, H-1’), 4.88 (d, 1H, J = 11.3 Hz, CHHPh), 4.90 (s, 1H, H-1), 4.95 (d, 1H, J = 11.3 Hz, CHHPh), 5.45 (t, 1H, J = 9.7 Hz, H-4’), 5.64 (s, 1H, CHPh), 6.84 (d, 2H, J = 9.0 Hz, H Arom pMP), 7.07 (d, 2H, J = 9.0 Hz, H Arom pMP), 7.10 – 7.60 (m, 20H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.5 (CH3 OMe), 55.6 (CH3 COOMe), 66.7 (C-5), 68.7 (C-4’), 68.9 (C-6), 71.6 (C-2’), 68.9 (CH2 Bn), 73.4 (CH2 Bn), 73.5 (C-5’), 75.2 (CH2 Bn), 75.7 (C-4), 77.7 (C-3), 79.0 (C-2, C-3’), 100.8 (CHPh), 102.5 (C-1’), 103.1 (C1), 114.5 (CH Arom pMP), 118.8 (CH Arom pMP), 126.3 – 128.8 (CH Arom), 137.7 (Cq Bn), 137.9 (Cq Bn), 138.2 (Cq Bn), 138.5 (Cq Bn), 151.4 (Cq pMP), 155.4 (Cq pMP), 167.9 (C=O Ac or COOMe), 169.5 (C=O COOMe or Ac); 13C-GATED (125 MHz, CDCl3): 102.5 (JC1’,H1’ = 164 Hz, C-1’), 103.1 (JC1,H1 = 158 Hz, C-1); HRMS: [M+NH4]+ calcd for C50H56O14N 894.37008, found 894.36879. 1,2;5,6-Di-O-isopropylidene-3-O-(methyl 4-O-acetyl-2,3-di-O-benzyl-β-D-mannopyranosyluronate)-α-D-glucopyranose (19): Donor 6 was glycosylated with acceptor 18 in the same way as described in the general procedure (Ph2SO/Tf2O) delivering the title compound 19 as a colorless oil. TLC: 30% EtOAc/PE; [α]D22:

O

MeOOC OBn O AcO BnO

O

O H O O

-34° (c = 0.7, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 1.22 (s, 3H, CH3 isoprop), 1.24 (s, 3H, CH3 isoprop), 1.35 (s, 3H, CH3 isoprop), 1.49 (s, 3H, CH3 isoprop), 1.96 (s, 3H, CH3 Ac), 3.42 (dd, 1H, J = 9.6 Hz, J = 3.2 Hz, H-3’), 3.65 (s, 3H, CH3 COOMe), 3.76 (d, 1H, J = 9.2 Hz, H-2’), 3.77 (d, 1H, J = 3.2 Hz, H-5’), 3.97 (dd, 1H, J = 8.4 Hz, J = 6.0 Hz, H-6), 4.09 (dd, 1H, J = 8.4 Hz, J = 6.8 Hz, H-6), 4.18 (d, 1H, J = 3.2 Hz, H-3), 4.25 (dd, 1H, J = 4.9 Hz, J = 3.2 Hz, H-4), 4.33 (d, 1H, J = O

12.0 Hz, CHHPh), 4.34 (d, 1H, J = 4.0 Hz, H-2), 4.39 (d, 1H, J = 12.0 Hz, CHHPh), 4.45 (dd, 1H, J = 11.2 Hz, J = 1.6 Hz, H-5), 4.46 (s, 1H, H-1’), 4.67 (d, 1H, J = 12.0 Hz, CHHPh), 4.76 (d, 1H, J = 12.4 Hz, CHHPh), 5.43 (t, 1H, J = 9.6 Hz, H-4’), 5.82 (d, 1H, J = 3.6 Hz, H-1), 7.15 – 7.32 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 20.0 (CH3 isoprop), 26.3 (CH3 isoprop), 26.5 (CH3 isoprop), 26.7 (CH3 isoprop), 52.6 (CH3 COOMe), 65.9 (C-6), 68.6 (C-3’), 71.8 (CH2 Bn), 73.3 (C-5’), 73.5, 73.6 (C-2’ and C-5), 73.9 (CH2 Bn), 78.2 (C-4’), 80.5 (C-4), 81.8 (C-3), 83.0 (C-2), 99.8 (C-1’), 104.9 (C-1), 108.4 (Cq isoprop), 111.9 (Cq isoprop), 127.5 – 128.4 (CH Arom), 137.6 (Cq Bn), 137.9 (Cq Bn), 167.7 (C=O Ac or COOMe), 169.5 (C=O Ac or COOMe); 13C-GATED (100 MHz, CDCl3): 99.8 (JC1’,H1’ = 157 Hz, C-1’), 104.9 (JC1,H1 = 181 Hz, C-1); HRMS: [M+H]+ calcd for C35H45O13 673.28547 found 673.28598.

MeOOC OBn O AcO BnO O BnO

COOMe O

OpMP

para-Methoxyphenyl (methyl 2,3-di-O-benzyl-4-O-(methyl 4-O-acetyl-2,3-diO-benzyl-α/β-D-mannopyranosyluronate)-β-D-galactopyranoside) uronate (21): Donor 6 was glycosylated with acceptor 20 in the same way as described in the general procedure (NIS/TMSOTf) delivering the title compound 21 as a

colorless oil. TLC: 30% EtOAc/PE; α-anomer: [α]D22: +2° (c = 0.3, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 2.03 (s, 3H, CH3 Ac), 3.53 (dd, 1H, J = 7.3 Hz, J = 3.1 Hz, H-3), 3.54 (s, 3H, CH3 COOMe), 3.57 (s, 3H, CH3 COOMe), 3.77 (s, 4H, H-2’, CH3 OMe), 3.86 (dd, 1H, J = 8.3 Hz, J = 3.0 Hz, H-3’), OBn

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Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    3.91 (dd, 1H, J = 9.8 Hz, J = 7.8 Hz, H-2), 4.09 (s, 1H, H-5), 4.53 (d, 1H, J = 12.3 Hz, CHHPh), 4.54 (d, 1H, J = 1.1 Hz, H-4), 4.61 (d, 1H, J = 12.3 Hz, CHHPh), 4.62 (d, 1H, J = 11.7 Hz, CHHPh), 4.65 (d, 1H, J = 10.9 Hz, CHHPh), 4.68 (d, 1H, J = 8.1 Hz, H-5’), 4.76 (d, 1H, J = 12.6 Hz, CHHPh), 4.80 (d, 1H, J = 7.7 Hz, H-1’), 4.83 (d, 1H, J = 12.6 Hz, CHHPh), 4.91 (d, 1H, J = 10.9 Hz, CHHPh), 5.01 (d, 1H, J = 10.9 Hz, CHHPh), 5.15 (d, 1H, J = 2.5 Hz, H-1’), 5.49 (t, 1H, J = 8.1 Hz, H-4’), 6.81 (d, 2H, J = 9.1 Hz, CH Arom pMP), 7.07 (d, 2H, J = 9.1 Hz, CH Arom pMP), 7.24 – 7.40 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.9 (CH3 Ac), 52.3 (CH3 COOMe), 52.4 (CH3 COOMe), 55.6 (CH3 OMe), 69.3 (C-4’), 71.2 (C-5’), 71.8 (CH2 Bn), 72.6 (CH2 Bn), 72.8 (CH2 Bn), 73.3 (C-4), 73.6 (C-2’), 75.2 (CH2 Bn), 75.3 (C-5), 76.4 (C-3’), 77.6 (C-2), 78.7 (C-3), 99.8 (C1’), 103.1 (C-1), 114.5 (CH Arom pMP), 118.8 (CH Arom pMP), 127.4 – 128.3 (CH Arom), 137.7 (Cq Bn), 138.0 (Cq Bn), 138.3 (Cq Bn), 151.6 (Cq pMP), 155.5 (Cq pMP), 167.7 (C=O COOMe or Ac), 169.0 (C=O COOMe or Ac), 169.9 (C=O COOMe or Ac); 13C-GATED (100 MHz, CDCl3): 99.8 (JC1’,H1’ = 168 Hz, C-1’), 103.1 (JC1,H1 = 160 Hz, C-1); HRMS: [M+NH4]+ calcd for C51H54O15 924.38010, found 924.38025. β-anomer: [α]D22: -34° (c = 0.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ = 1.99 (s, 3H, CH3 Ac), 3.24 (dd, 1H, J = 9.8 Hz, J = 2.8 Hz, H-3’), 3.63 (dd, 1H, J = 9.6 Hz, J = 3.0 Hz, H-3), 3.67 (d, 1H, J = 9.9 Hz, H-5’), 3.72 (s, 3H, CH3 COOMe), 3.77 (s, 3H, CH3 OMe), 3.79 (s, 3H, CH3 COOMe), 3.93 (d, 1H, J = 2.9 Hz, H-2’), 3.95 (dd, 1H, J = 9.6 Hz, J = 7.7 Hz, H-2), 4.15 (d, 1H, J = 1.1 Hz, H-5), 4.25 (d, 1H, J = 12.3 Hz, CHHPh), 4.86 (d, 1H, J = 12.3 Hz, CHHPh), 4.53 (dd, 1H, J = 3.0 Hz, J = 1.1 Hz, H-4), 4.62 (d, 1H, J = 11.7 Hz, CHHPh), 4.76 (d, 1H, J = 11.7 Hz, CHHPh), 4.77 (d, 1H, J = 2.9 Hz, H-1’), 4.78 (d, 1H, J = 10.9 Hz, CHHPh), 4.84 (d, 1H, J = 7.7 Hz, H-1), 4.86 (d, 1H, J = 12.6 Hz, CHHPh), 4.95 (d, 1H, J = 12.6 Hz, CHHPh), 5.05 (d, 1H, J = 10.9 Hz, CHHPh), 5.41 (t, 1H, J = 9.8 Hz, H-4’), 6.82 (d, 2H, J = 9.1 Hz, CH Arom pMP), 7.10 (d, 2H, J = 9.1 Hz, CH Arom pMP), 7.23 – 7.54 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.4 (CH3 COOMe), 52.6 (CH3 COOMe), 55.6 (CH3 OMe), 68.6 (C-4’), 71.1 (CH2 Bn), 71.7 (C-2’), 73.1 (C-4), 73.3 (CH2 Bn), 73.5 (CH2 Bn), 73.7 (C-5’), 73.7 (C-5), 75.3 (CH2 Bn), 78.5 (C-2), 78.6 (C-3’), 80.9 (C-3), 101.4 (C-1’), 103.2 (C-1), 114.5 (CH Arom pMP), 119.3 (CH Arom pMP), 127.3 – 128.8 (CH Arom), 137.7 (Cq Bn), 137.9 (Cq Bn), 138.3 (Cq Bn), 138.6 (Cq Bn), 151.4 (Cq pMP), 155.6 (Cq pMP), 167.1 (C=O COOMe or Ac), 167.9 (C=O COOMe or Ac), 169.6 (C=O COOMe or Ac); 13C-GATED (100 MHz, CDCl3): 101.4 (JC1’,H1’ = 160 Hz, C-1’), 103.2 (JC1,H1 = 160 Hz, C-1); HRMS: [M+Na]+ calcd for C51H54O15Na 929.33549, found 929.33706. Methyl (phenyl 4-O-levulinoyl-2,3-di-O-benzyl-1-thio-α-D-mannopyranoside) uronate (22): Compound 5 (276 mg g, 0.57 mmol) was dissolved in 5.7 mL pyridine and levulinoyl SPh anhydride (0.5M in dioxane, 3.4 mL, 1.7 mmol) was added. The reaction was stirred overnight, after which the mixture was diluted with EtOAc, washed with 1M HCl, saturated aqueous NaHCO3 and brine. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude MeOOC OBn O LevO BnO

product was purified by flash column chromatography (20% EtOAc/PE) to yield 330 mg of the title compound 22 (0.57 mmol, 86%) as a colorless oil. TLC: 30% EtOAc/PE; 1H NMR (400 MHz, CDCl3) δ = 2.17 (s, 3H, CH3 Lev), 2.52 (m, 2H, CH2 Lev), 2.69 – 2.72 (m, 2H, CH2 Lev), 3.58 (s, 3H, CH3 COOMe), 3.74 (bd, 1H, J = 6.0 Hz, H-2), 3.86 (dd, 1H, J = 6.0 Hz, J = 2.8 Hz, H-3), 4.53 (d, 1H, J = 12.0 Hz, CHHPh), 4.54 (s, 1H, H-5), 4.56 (d, 1H, J = 12.8 Hz, CHHPh), 4.61 (d, 1H, J = 12.8 Hz, CHHPh), 4.64 (d, 1H, J = 12.0 Hz, CHHPh), 5.57 (t, 1H, J = 5.6 Hz, H-4), 5.78 (d, 1H, J = 7.2 Hz, H-1), 7.21 – 7.60 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 27.8 (CH2 Lev), 29.7 (CH3 Lev), 37.7 (CH2 Lev), 52.2 (CH3 COOMe), 69.6 (C-4), 72.9 (C-5), 73.8 (C-3), 74.3 (C-2), 82.9 (C-1), 168.4 (C=O Lev or COOMe), 171.5 (C=O COOMe or Lev), 206.0 (C=O Lev ketone); HRMS: [M+NH4]+ calcd for C32H38O8SN 596.23126, found 596.23024.

77   

77

Chapter 4    MeOOC OBn O AcO BnO

3-Azidopropyl (methyl (4-O-acetyl-2,3-di-O-benzyl-β-D-mannopyranoside) uronate) (23): A solution of 102 mg donor 6 (0.2 mmol 1 equiv.), 51 mg diphenyl sulfoxide (0.25 mmol, 1.3 equiv.) and 124 mg tri-tert-butylpyrimidine (0.5 mmol, 2.5 equiv.) in 5 mL DCM was stirred over activated MS3Å for 30min. The mixture was cooled to -60°C before O

N3

41 µL triflic acid anhydride (0.25 mmol, 1.3 equiv.) was added. The mixture was allowed to warm to -40°C in 15min followed by cooling to -80°C.24 At that temperature 30 mg acceptor (0.3 mmol, 1.5 equiv.) in 1 mL DCM was added. Stirring was continued and the reaction mixture was allowed to warm to rT followed by addition of 2 mL pyridine and 1 mL Ac2O. Stirring was continued for a further 10h. After addition of MeOH, the reaction mixture was diluted with EtOAc and washed with 1M HCl and NaHCO3 (sat.). The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded 80 mg of title compound 23 (α/β = 1/5, 0.16 mmol, 80%) as a colorless oil. TLC: 50% EtOAc/PE; [α]D22: -2° (c = 0.4, CHCl3); IR (neat, cm-1): 1091, 1211, 1265, 1450, 1732, 2110; 1H NMR (400 MHz, CDCl3) δ = 1.84 – 1.96 (m, 2H, CH2 azidopropyl), 2.02 (s, 3H, CH3 Ac), 3.39 (t, 2H, CH2 azidopropyl), 3.48 – 3.56 (m, 2H, H-3, CH azidopropyl), 3.72 (s, 3H, CH3 COOMe), 3.85 (d, 1H, J = 9.6 Hz, H-5), 3.89 (d, 1H, J = 2.0 Hz, H-2), 4.01 – 4.07 (m, 1H, CH2 azidopropyl), 4.38 (d, 1H, J = 12.4 Hz, CHHPh), 4.44 (s, 1H, H1), 4.50 (d, 1H, J = 12.4 Hz, CHHPh), 4.80 (d, 1H, J = 12.4 Hz, CHHPh), 4.92 (d, 1H, J = 12.4 Hz, CHHPh), 5.52 (t, 1H, J = 9.6 Hz, H-4), 7.21 – 7.42 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 29.0 (CH2 azidopropyl), 48.3 (CH2 azidopropyl), 52.6 (CH3 COOMe), 66.9 (CH2 azidopropyl), 68.9 (C-4), 71.5 (CH2 Bn), 73.0 (C-2), 73.7 (C-5), 73.8 (CH2 Bn), 78.1 (C-3), 101.5 (C-1), 127.4 – 128.3 (CH Arom), 137.7 (Cq Bn), 138.2 (Cq Bn), 167.9 (C=O Ac), 169.6 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 101.5 (J = 154 Hz, C-1); HRMS: [M+Na]+ calcd for C26H31O8N3Na 536.20034, found 536.20156. MeOOC OBn O HO BnO

3-Azidopropyl (methyl 2,3-di-O-benzyl-β-D-mannopyranoside) uronate (24): To a

stirred solution of 80 mg compound 23 (1.6 mmol) in MeOH was added a catalytic N3 amount of sodium methoxide (30% wt in MeOH). The mixture was stirred for 6h before Amberlite IR120+ resin was added to acidify the solution to pH = 6. Subsequent filtration and concentration under reduced pressure afforded the crude product. Flash chromatography (EtOAc/PE) and removal of the eluent afforded 44 mg of title compound 24 (β-isomer was selectively hydrolyzed, 60%) as a O

colorless oil. TLC: 50% EtOAc/PE; [α]D22: -21° (c = 0.2, CHCl3); IR (neat, cm-1): 1091, 1211, 1265, 1450, 1732, 2110; 1H NMR (400 MHz, CDCl3) δ = 1.89 – 2.02 (m, 2H, CH2 azidopropyl), 3.02 (s, 1H, OH-4), 3.39 (dd, 1H, J = 9.5 Hz, J = 2.9 Hz, H-3), 3.44 (t, 2H, J = 6.7 Hz, CH2 azidopropyl), 3.57 – 3.66 (m, 1H, CH azidopropyl), 3.80 (d, 1H, J = 9.6 Hz, H-5), 3.85 (s, 3H, CH3 COOMe), 3.92 (d, 1H, J = 2.7 Hz, H-2), 4.06 – 4.11 (m, 1H, CH azidopropyl), 4.34 (t, 1H, J = 9.6 Hz, H-4), 4.47 (s, 1H, H-1), 4.55 (d, 1H, J = 12.0 Hz, CHHPh), 4.61 (d, 1H, J = 12.0 Hz, CHHPh), 4.81 (d, 1H, J = 12.4 Hz, CHHPh), 4.97 (d, 1H, J = 12.4 Hz, CHHPh), 7.30 – 7.47 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 28.9 (CH2 azidopropyl), 48.2 (CH2 azidopropyl), 52.5 (CH3 COOMe), 66.8 (CH2 azidopropyl), 68.2 (C-4), 71.7 (CH2 Bn), 73.4 (C-2), 74.1 (CH2 Bn), 74.9 (C-5), 80.2 (C-3), 101.9 (C-1), 127.4 – 128.3 (CH Arom), 137.7 (Cq Bn), 138.2 (Cq Bn), 169.6 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 101.9 (J = 153 Hz, C-1); HRMS: [M+Na]+ calcd for C24H29O7N3Na 494.18977, found 494.19008. MeOOC OBn O

LevO BnO

MeOOC OBn O O BnO

O N3

3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl 4-O-levulinoyl2,3-di-O-benzyl-β-D-mannopyranosyl-uronate)-β-D-mannopyranoside) uronate (25): Disaccharide 25 was obtained from donor 22 and

acceptor 24 according to method A of the general procedure for glycosidations using NIS/TMSOTf giving 73 mg title compound 25 (0.078 mmol, 78%) as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: -26° (c = 1.0, CHCl3); IR (neat, cm-1): 1024, 1053, 1361, 1454, 1496, 1747, 2110, 2869;

78   

78

Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters    H NMR (400 MHz, CDCl3) δ = 1.83 – 1.89 (m, 2H, CH2 azidopropyl), 2.14 (CH3 Lev), 2.50 (q, 2H, J = 6.0 Hz, CH2 Lev), 2.67 (t, 2H, J = 6.0 Hz, CH2 Lev), 3.34 (t, 1H, J = 6.8 Hz, CH2 azidopropyl), 3.45 (dd, 1H, J = 9.6 Hz, J = 2.8 Hz, H-3’), 3.49 – 3.54 (m, 1H, CH azidopropyl), 3.54 (s, 3H, CH3 COOMe), 3.60 (dd, 1H, J = 8.8 Hz, J = 3.2 Hz, H-3), 3.62 (s, 3H, CH3 COOMe), 3.68 (d, 1H, J = 10.0 Hz, H-5’), 3.82 (bs, 2H, H-2, H-2’), 3.87 (d,

1

1H, J = 8.8 Hz, H-5), 4.98 – 4.02 (m, 1H, CH azidopropyl), 4.39 (t, 1H, J = 8.8 Hz, H-4), 4.42 (d, 1H, J = 12.0 Hz, CHHPh), 4.46 (s, 1H, H-1), 4.52 (d, 1H, J = 12.0 Hz, CHHPh), 4.56 (d, 1H, J = 12.4 Hz, CHHPh), 4.69 (s, 1H, H-1’), 4.70 (d, 1H, J = 12.8 Hz, CHHPh), 4.83 (d, 1H, J = 12.4 Hz, CHHPh), 4.78 (d, 1H, J = 12.8 Hz, CHHPh), 4.83 (d, 1H, J = 12.0 Hz, CHHPh), 5.42 (t, 1H, J = 9.6 Hz, H-4’), 7.22 – 7.38 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 27.8 (CH2 Lev), 29.0 (CH2 azidopropyl), 29.8 (CH3 Lev), 37.7 (CH2 Lev), 48.2 (CH2 azidopropyl), 52.3 (CH3 COOMe), 66.8 (CH2 azidopropyl), 68.9 (C-4’), 71.7 (CH2 Bn), 72.2 (CH2 Bn), 73.3 (C-5’), 73.9 (CH2 Bn), 73.9 (C-2 or C-2’), 74.3 (CH2 Bn), 74.3 (C-5), 74.9 (C-2’ or C-2), 77.5 (C-4), 78.5 (C-3’), 79.3 (C-3), 101.8 (C-1), 102.3 (C-1’), 127.1 – 128.3 (CH Arom), 137.8 (Cq Bn), 138.3 (Cq Bn), 138.6 (Cq Bn), 138.6 (Cq Bn), 167.7 (C=O COOMe), 168.6 (C=O COOMe), 171.6 (C=O Lev ester), 206.1 (C=O Lev ketone); 13C-GATED (100 MHz, CDCl3): 101.8 (JC1,H1 = 155 Hz, C-1), 102.3 (JC1’,H1’ = 156 Hz, C-1’); HRMS: [M+Na]+ calcd for C50H57O15N3Na 962.36819, found 962.36725. MeOOC OBn O

HO BnO

MeOOC OBn O O BnO

3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl 2,3-di-O-benzylβ-D-mannopyranosyluronate)-β-D-mannopyranoside) uronate (26):

O

Disaccharide 25 (65 mg, 0.069 mmol) was dissolved in a mixture of pyridine and acetic acid (1 mL, 4/1 v/v), after which 17 µL hydrazine monohydrate (0.35 mmol) was added. The mixture was stirred for 30min and then diluted with EtOAc, washed with 2M HCl and satured NaHCO3. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/hexane) afforded 55 mg of the title compound 26 (0.66 N3

mmol, 95%) as a colorless oil. TLC: 20% EtOAc/DCM; [α]D22: +35.6° (c = 0.7, CHCl3); IR (neat, cm-1): 1026, 1053, 1361, 1454, 1496, 1747, 2094, 2869; 1H NMR (400 MHz, CDCl3) δ = 1.82 – 1.94 (m, 2H, CH2 azidopropyl), 2.94 (bs, 1H, OH-4’), 3.31 (dd, 1H, J = 9.6 Hz, J = 3.8 Hz, H-3’), 3.35 (t, 2H, J = 7.0 Hz, CH2 azidopropyl), 3.51 – 3.55 (m, 1H, CH azidopropyl), 3.59 (d, 1H, J = 9.6 Hz, H-5’), 3.62 (s, 3H, CH3 COOMe), 3.64 (s, 3H, CH3 COOMe), 3.65 (dd, 1H, J = 8.8 Hz, J = 2.8 Hz, H-3), 3.83 (d, 1H, J = 2.8 Hz, H-2’), 3.86 (d, 1H, J = 2.8 Hz, H-2), 3.90 (d, 1H, J = 8.8 Hz. H-5), 4.00 – 4.06 (m, 1H, CH azidopropyl), 4.19 (t, 1H, J = 9.6 Hz, H-4’), 4.45 (t, 1H, J = 8.6 Hz, H-4), 4.48 (s, 1H, H-1), 4.56 (d, 1H, J = 12.0 Hz, CHHPh), 4.57 (d, 1H, J = 12.4 Hz, CHHPh), 4.60 (d, 1H, J = 12.4 Hz, CHHPh), 4.68 (d, 1H, J = 12.8 Hz, CHHPh), 4.73 (s, 1H, H-1’), 4.74 (d, 1H, J = 12.0 Hz, CHHPh), 4.76 (d, 1H, J = 12.0 Hz, CHHPh), 4.78 (d, 1H, J = 12.8 Hz, CHHPh), 4.83 (d, 1H, J = 12.0 Hz, CHHPh), 7.23 – 7.38 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 29.0 (CH2 azidopropyl), 48.2 (CH2 azidopropyl), 52.3 (CH3 COOMe), 52.4 (CH3 COOMe), 66.8 (CH2 azidopropyl), 68.1 (C-4’), 71.8 (CH2 Bn), 72.1 (CH2 Bn), 73.9 (CH2 Bn), 73.9 (C-2), 74.5 (C-5), 74.6 (CH2 Bn), 74.8 (C-5’), 75.2 (C-2’), 77.2 (C-4), 79.3 (C-5’), 80.4 (C-3’), 101.7 (C-1), 102.4 (C-1’), 127.0 – 128.4 (CH Arom), 137.9 (Cq Bn), 138.3 (Cq Bn), 138.6 (Cq Bn), 138.8 (Cq Bn), 168.6 (C=O COOMe), 169.8 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 101.7 (JC1,H1 = 154 Hz, C-1), 102.4 (JC1’,H1’ = 155 Hz, C-1’); ESI-MS: 864.3 [M+Na]+. MeOOC OBn O

LevO BnO

MeOOC OBn O O BnO

MeOOC OBn O O BnO

O N3

3-Azidopropyl (methyl 2,3-di-O-benzyl-4-O-(methyl 2,3-di-O-benzyl-4-O-(methyl 4-O-levulinoyl-2,3-di-Obenzyl-β-D-mannopyranosyluronate)-β-D-mannopyranosyl) uronate)-β-D-mannopyranoside) uronate (27): Trisaccharide 27 was obtained from donor 22 and acceptor 26 according to method A of the general procedure

79   

79

Chapter 4    for glycosidations using NIS/TMSOTf giving 33 mg title compound 27 (α/β = 1/>10), 0.026 mmol, 70%) as a colorless oil. TLC: 30% EtOAc/PE; IR (neat, cm-1): 1091, 1211, 1265, 1450, 1732, 2110; 1H NMR (600 MHz, CDCl3) δ = 1.82 – 1.90 (m, 2H, CH2 azidopropyl), 2.14 (s, 3H, CH3 Lev), 2.52 (q, 2H, J = 7.2 Hz, J = 6.6 Hz, CH2 Lev), 2.67 (t, 2H, J = 6.6 Hz, CH2 Lev), 3.34 (t, 2H, CH2 azidopropyl), 3.41 (dd, 1H, J = 9.6 Hz, J = 2.4 Hz, H-3’’), 3.49 (dd, 1H, J = 9.0 Hz, J = 3.3 Hz, H-3), 3.45 (s, 4H, CH azidopropyl and CH3 COOMe), 3.56 – 3.60 (m, 4H, H-3’ and CH3 COOMe), 3.65 (d, 1H, J = 9.6 Hz, H-5’’), 3.74 (d, 1H, J = 2.4 Hz, H-2), 3.76 (d, 1H, J = 9.6 Hz, H-5), 3.79 (d, 1H, J = 2.4 Hz, H-2’’), 3.81 (d, 1H, J = 1.8 Hz, H-2’), 3.82 (d, 1H, J = 8.4 Hz, H-5’), 4.00 – 4.03 (m, 1H, J = 6.1 Hz, J = 5.4 Hz, CH azidopropyl), 4.30 (t, 1H, J = 9.6 Hz, H-4), 4.41 (d, 1H, J = 12.0 Hz, CHHPh), 4.42 (t, 1H, J = 9.4 Hz, H-4’), 4.44 (s, 1H, H-1), 4.47 (d, 1H, J = 12.0 Hz, CHHPh), 4.53 (d, 1H, J = 12.0 Hz, CHHPh), 4.56 (d, 1H, J = 12.0 Hz, CHHPh), 4.59 (s, 1H, H-1’’), 4.60 (d, 1H, J = 12.0 Hz, CHHPh), 4.65 (d, 1H, J = 12.0 Hz, CHHPh), 4.71 (s, 1H, H-1’), 4.75 (d, 1H, J = 12.0 Hz, CHHPh), 4.77 (d, 1H, J = 12.0 Hz, CHHPh), 4.79 (d, 1H, J = 12.0 Hz, CHHPh), 4.79 (d, 1H, J = 12.0 Hz, CHHPh), 4.81 (d, 1H, J = 12.0 Hz, CHHPh), 4.83 (d, 1H, J = 12.0 Hz, CHHPh), 5.40 (t, 1H, J = 9.6 Hz, H-4’’), 7.16 – 7.36 (m, 30H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 27.8 (CH2 Lev), 29.0 (CH2 azidopropyl), 29.8 (CH3 Lev), 37.8 (CH2 Lev), 48.2 (CH2 azidopropyl), 52.1 (CH3 COOMe), 52.3 (CH3 COOMe), 52.4 (CH3 COOMe), 66.8 (CH2 azidopropyl), 68.9 (C-4’), 71.6 (CH2 Bn), 72.3 (CH2 Bn), 72.5 (CH2 Bn), 73.3 (C-5’’), 73.8 (CH2 Bn), 74.1 (C-2’), 74.3 (CH2 Bn), 74.4 (C-5), 74.5 (C-5’), 74.5 (CH2 Bn), 74.9 (C-2’’), 75.9 (C-2), 77.1 (C-4’), 77.7 (C-4’’), 78.4 (C-3’’), 79.3 (C3’), 79.6 (C-3), 101.7 (C-1), 102.4 (C-1’’), 102.5 (C-1’), 127.2 – 128.4 (CH Arom), 137.8 (Cq Bn), 138.4 (Cq Bn), 138.7 (Cq Bn), 138.7 (Cq Bn), 138.8 (Cq Bn), 167.8 (C=O COOMe or Lev), 168.6 (C=O COOMe or Lev), 168.6 (C=O COOMe or Lev), 171.6 (C=O COOMe or Lev), 206.2 (C=O Lev ketone); 13C-GATED (150 MHz, CDCl3): 101.6 (JC1’,H1’ = 155 Hz, C-1’), 102.4 (JC1,H1 = 158 Hz, C-1), 102.5 (JC1’’,H1’’ = 156 Hz, C-1’’); HRMS: [M+Na]+ calcd for C71H79O21N3Na 1332.50983, found 1332.51029. HOOC OH O

HO HO

HOOC OH O O HO

HOOC OH O O HO

O NH2

3-Aminopropyl (4-O-(4-O-(β-D-mannopyranosyluronate)-β-D-mannopyranosyluronate)-β-D-mannopyranoside) uronic acid (28): Trisaccharide 27 (35mg, 0.027mmol) was dissolved in 0.5 mL pyridine/AcOH (4/1) before 5 µL N2H4·H2O (0.1mmol) was added. The mixture was allowed to stir for 10min, diluted with EtOAc and subsequently washed with 2M HCl and sat NaHCO3. The organic phase is dried (MgSO4), filtered and concentrated under reduced pressure. The crude intermediate was dissolved in 1 mL THF/H2O and treated with 0.15 mL KOH (0.45M) for 1h. The reaction mixture was neutralized with Amberlite IR120 to pH = 3, filtered and concentrated under reduced pressure. The residual oil is dissolved in 1 mL MeOH/AcoH (10/1) before 25 mg Pd/C (10%) is added. The reaction mixture is stirred overnight under an H2-atmosphere followed by filtration. Gel filtration (HW-40, 0.15M Et4NOAc in H2O) of the residual oil afforded the desired trisaccharide 28 (5 mg, 8.3 µmol, 35% over 3 steps) as a white foam. TLC: 50% iPrOH/H2O; 1H NMR (400 MHz, CDCl3) δ = 1.99 – 2.03 (m, 2H, CH2 spacer), 3.14 – 3.21 (m, 2H, CH2 spacer), 3.38 (s, 1H), 3.67 – 3.70 (m, 2H), 3.74 – 3.78 (m, 1H), 3.79 – 3.84 (m, 3H), 3.86 – 3.90 (m, 1H), 3.89 – 3.93 (m, 2H), 3.97 – 4.03 (m, 2H), 4.05 – 4.07 (m, 2H), 4.66 (s, 1H), 4.69 (s, 1H), 4.77 (s, 1H); 13C NMR (100 MHz, CDCl3) δ = 27.5, 38.5, 63.6, 67.8, 68.4, 69.3, 70.1, 70.6, 70.8, 71.2, 72.3, 73.4, 75.7, 76.7, 76.8, 100.7, 100.8, 101.0, 176.1, 176.4, 177.0; ESI-MS: 629.7 [M+H]+.

80   

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Stereocontrolled Synthesis of β‐D‐Mannuronic Acid Esters   

References and Notes 1 2 3

Original publication: Van den Bos, L.J.; Dinkelaar, J.; Overkleeft, H.S.; Van der Marel, G.A. J. Am. Chem. Soc. 2006, 128, 13066–13067. Moe, S.T.; Draget, K.I.; Skjak-Braek, G.; Smidsrød, O. In Food Polysaccharides and Their Applications, Stephen A.M. ed., Marcel Dekker, Inc., New York, 1995, 245–286. Alginates are officially approved in the class of emulsifiers, stabilizers, thickeners and gelling agents (E400 – E404). http://www.food.gov.uk/safereating/chemsafe/additivesbranch/enumberlist#h_6.

4

Flo, T.H.; Ryan, L.; Latz, E.; Takeuchi, O.; Monks, B.G.; Lien, E.; Halaas, O.; Akira, S.; Skjak-Braek, G.; Golenbock, D.T.; Espevik, T.J. J. Biol. Chem. 2002, 277, 35489–35495. Iwamoto, M.; Kurachi, M.; Nakashima, T.; Kim, D.; Yamaguchi, K.; Oda, T.; Iwamoto, Y.; Muramatsu, T.I. FEBS Letters 2005, 579, 4423–4429.

5

7

(a) Janeway, C.A.; Medzhitov, R. Ann. Rev. Immunol. 2002, 20, 197–216; (b) Rich, T. Toll and Toll-Like Receptors An Immunologic Perspective, Kluwer Academic/Plenum Publishers: New York, 2005. (a) Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348; (b) Crich, D.; Sun, S. J. Am. Chem. Soc. 1998, 120, 435–436; (c) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. For a review on β-mannoside synthesis: El Ashry, E.S.H.; Rashed, N.; Ibrahim, E.S.I. Curr. Org. Synth.

8 9 10 11

2005, 2, 175–213. Crich, D.; Chandrasekera, N.S. Angew. Chem. Int. Ed. 2004, 43, 5386–5389. See Chapter 5 for more details. Jensen, H.H.; Nordstrøm, L.U.; Bols, M. J. Am. Chem. Soc. 2004, 126, 9205–9213. Dean, K.E.S.; Kirby, A.J.; Komarov, I.V. J. Chem. Soc., Perkin Trans. 2 2002, 337–341.

6

12 (a) Srivastava, V.K.; Schuerch, C. Carbohydr. Res. 1980, 79, C13–C16; (b) Srivastava, V.K.; Schuerch, C. J. Org. Chem. 1981, 46, 1121–1126. (c) Crich, D.; Hutton, T.K.; Banerjee, A.; Jayalath, P.; Picione, J. Tetrahedron Asymm. 2005, 16, 105–119. 13 (a) see Chapter 2 (b) Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168. 14 (a) see Chapter 3 (b) Van den Bos, L.J.; Litjens, R.E.J.N.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2005, 7, 2007–2010. (c) Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950. 15 Crich, D.; Smith, M. J. Am. Chem. Soc. 2002, 124, 8867–8869. 16 Zuurmond, H.M.; Van der Klein, P.A.M.; Van der Marel, G.A.; Van Boom, J.H. Tetrahedron 1993, 49, 6501–6514. 17 Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 2, 3797–3800. 18 The anomeric configurations were determined by measuring 13C-GATED NMR spectra: Bock, K.; Pedersen, C. J. Chem. Soc., Perk. Trans. 2 1974, 293–299. 19 Recently, Demchenko and coworkers proposed the possibility of remote participation from the C4 position: De Meo, C.; Kamat, M.N.; Demchenko, A.V. Eur. J. Org. Chem. 2005, 706–711. 20 (a) Crich, D.; Cai, W.; Dai, Z. J. Org. Chem. 2000, 65, 1291–1297 (b) See Chapter 9. 21 This is in accord with Paulsen who stated that primary alcohol acceptors are considerably more reactive and tend to react with less anomeric selectivity than their secondary counterparts. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155–173. 22 Veeneman, G.H.; Van Leeuwen, S.H.; Van Boom, J.H. Tetrahedron Lett. 1990, 31, 1331–1334.

81   

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Chapter 4    23 (a) Crich, D.; De la Mora, M.; Vinod, A.U. J. Org. Chem. 2003, 68, 8142–8148. (b) Codée, J.D.C.; Stubba, B.; Schiattarella, M.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. J. Am. Chem. Soc. 2005, 127, 3767–3773. 24 Addition of the 3-azidopropanol acceptor to the activated reaction mixture at -80°C substantially increases βselectivity compared to adding the acceptor at -50°C (α/β = 1/1, 76%). 25 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326. 26 Assignment was complicated due to low peak intensities in the 13C-APT spectrum.

82   

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Chapter 5 │  Study of the Glycosylation     Properties of Deactivated 1‐Thio    Mannosazidopyranosides

Abstract: A study of the glycosylation properties of 1-thiomannosazides with a variety of electron withdrawing substituents is presented. The assembly of the fully protected trisaccharide 24, corresponding to the repeating unit of the enterobacterial common antigen, using both an orthogonal and a chemoselective coupling strategy is described.1  

Introduction N-Acetyl mannosamine and N-acetyl mannosaminuronic acids are constituents of various bacterial capsular polysaccharides and lipopolysaccharides.2 In most cases, these mannoside residues are connected to the next monosaccharide residue via a 1,2-cis linkage.3 Viewing along the C2-C1 bond shows that in the β-oriented linkage, the O2, O1 and O(ring) atoms are in close proximity (Δ2-condition), resulting in increased conformational instability as compared to the α-linked glycoside.4 Several methodologies for the synthesis of β-linked mannopyranoside residues have been explored.5 These include methods in which the O2 position of β-D-glucopyranosides is oxidized and reduced6 or displaced in an SN2-type fashion by an azido nucleophile.7 Other procedures involve silver silicate-mediated SN2-displacement of α-bromides8 or intramolecular aglycon delivery9 as the key step. A breakthrough in this field of research was reported by Crich and coworkers who discovered that activation of 4,6O-benzylidene protected mannopyranosyl sulfoxide donors with triflic anhydride leads to the formation of β-mannoside linkages.10 Later studies demonstrated that comparable β83   

83

Chapter 5   

selectivities are attained using 4,6-O-benzylidene protected 1-thiomannosides in combination with benzenesulfenyl triflate11 (PhSOTf) or 1-benzenesulfinyl piperidine (BSP)/triflic anhydride (Tf2O) as activation systems.12 The β-directing effect of the 4,6-O-benzylidene protective group was initially explained by increased torsional strain.13 Recently, Bols and coworkers argued that electronic effects14 have a major influence on the stereochemical outcome of glycosylations involving 4,6-O-benzylidene mannoside donors.15 The decisive influence of the 4,6-O-benzylidene protection in mannopyranose donors is further underscored by several other reports showing that variations in the glycosylation procedures and promotor systems only led to minor fluctuations in the stereochemical outcome of the βmannosylation reaction.16 Similarly, Litjens et al. observed stereoselective 1,2-cis glycosylation of 4,6-O-benzylidene protected 1-thio-α-D-mannosazidopyranoside donors using diphenyl sulfoxide (Ph2SO)/Tf2O17 as the activation system.18 The finding that the stereodirecting effect of a 4,6-O-benzylidene protective group on glycosyl transfer is at least partially governed by electronic effects suggests that the introduction of sufficiently electron withdrawing functionalities in the mannose core may also lead to β-selective mannosylation.19 In fact, Schuerch and coworkers demonstrated in 1980 that reaction of mannopyranose donors having a strongly electronegative non-participating substituent at C2 (such as tosyl) and a reactive, electronegative leaving group at C1 (such as tosyl or chloride) results in high β-selectivities.20 Application of this approach is limited by the harsh conditions required for removal of the C2-tosyl functionality and the moderate selectivities obtained with sterically hindered acceptor nucleophiles. Chapter 4 describes that the strong stereodirecting influence of carboxylic acid functionalized 1-thiomannosides favors SN2-type displacement of the putative α-triflate intermediate resulting in high to exclusive βselectivities.21 In addition to these studies, this Chapter describes the glycosylation properties in terms of yield and stereoselectivity of the 1-thio-α-D-mannosazidopyranoside donors 2, 5 and 8, functionalized with electron-withdrawing substituents at different positions of the pyranoside core. Next to the outcome of these studies, their application in the assembly of the fully protected repeating unit trisaccharide 24 of the enterobacterial common antigen (ECA) is presented.

Results and Discussion Donor glycosides 2, 5 and 8 were obtained from commercially available D-mannosamine hydrochloride 1 as described in Scheme 1. Subjection of 1 to diazo transfer, followed by acetylation and finally Lewis acid catalyzed introduction of the anomeric thiophenyl group gave tri-O-acetyl mannosazide 2.22 Deacetylation of compound 2 followed by installation of the 4,6-O-benzylidene acetal, introduction of the benzyl group at the C3 position and acidic removal of the benzylidene acetal yielded diol 4 in good yield. Acetylation of diol 4 afforded 84   

84

Deactivated 1‐Thiomannosazidopyranosides   

O3 benzylated glycoside 5. Chemo- and regioselective oxidation of the diol 4 using the TEMPO/BAIB reagent combination23 afforded the corresponding mannosaziduronic acid 6 in 65% yield. Methylation of the free carboxylic acid in 6 and acetylation of the residual C4 position afforded uronic ester donor 8.

Scheme 1. HO HO HO

NH2·HCl O

ref. 22

A cO AcO AcO

N3 O

OH 1

2

N3 O

1

R O 2 RO BnO

ref. 22

S Ph b

2

R O B nO

fro m 4

SPh 3: R =R =CHPh 1 2 4: R =R =H 1 2 5: R =R =Ac 1

a

1 R OOC N 3 O

c

2

d b

S Ph 6 : R1=R2=H 1 7 : R =Me, R 2=H 1 2 8 : R =Me, R =A c

Reagents and conditions: (a) TsOH, MeOH (b) Ac2O, pyridine, 7: 88% (from 2), 8: quant. (c) TEMPO, BAIB, DCM/H2O (4/1), 65% (from 4) (d) MeI, K2CO3, DMF, 71%.

With the target donor mannosides in hand, attention was focused on the investigation of their glycosylation properties. Phenyl 2-azido-3-O-benzyl-4,6-di-O-acetyl-2-deoxy-1-thio-αD-mannopyranoside (5) was condensed with methyl 2,3,4-tri-O-benzyl-α-D-glucopyranoside 9,24 under the influence of in situ generated diphenylsulfonium bistriflate (Table 1, entry 1). 2,4,6-Tri-tert-butylpyrimidine (TTBP) was added as acid scavenger.25 Within 15min, the temperature was gradually raised to -50°C followed by addition of acceptor 9 and further warming of the reaction mixture to room temperature. Disaccharide 10 was isolated in 86% yield as an anomeric mixture (α/β = 1/4).26 The outcome of this glycosylation in terms of yield and anomeric ratio compares favorably to the condensation of 4,6-O-benzylidene protected donor 3 with the same acceptor (entry 2).18a Condensation of di-acetyl donor 7 with the secondary alcohol acceptor 12, executed under the conditions described above gave disaccharide 13 in good yield and with a slight preference for α-anomer formation (entry 3). This decrease in β-selectivity agrees with the reported trend that glycosylation of a specific donor with a series of acceptors of decreasing nucleophilicity leads to a change of the anomeric ratio in favor of the α-product.27 Next, the reactivity of the mannosazide donor was further reduced by replacement of the 3-O-benzyl with the 3-O-acetyl protective group (2). Phenyl 2-azido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-α-D-mannopyranoside (2) was effectively condensed with the highly reactive 9, the less reactive 15 and the sterically demanding acceptor 12 to give disaccharides 14, 16 and 17, respectively (entries 4, 5 and 6). In all cases only the α-product was isolated suggesting that the stereochemical outcome of these reactions is independent of the reactivity of the acceptor. These results support earlier observations that 3-O-acyl groups in mannopyranose28 and glucopyranose29 donors are α-directing by neighboring group participation, which proceeds via a 6-membered transition state shielding β-site attack.30 Finally, the glycosylation properties of 1-thio-α-mannosaziduronic acid donor

85   

85

Chapter 5    Table 1. entry

1

donor AcO AcO BnO

218a

O

BnO BnO

BnO

S Ph

9

Ph2SO/Tf2O 86% (1/4)

O BnO

Ph2SO/Tf2O 77% (1/3)

5

O

Op MP

Ph2SO/Tf2O 82% (3/2)

AcO AcO BnO

N3 O

O

O

N3 O

AcO AcO AcO

Ph2SO/Tf2O 88% (1/0)

9

O OBn B nO

14

O

O

O

2

AcO AcO AcO

Ph2SO/Tf2O 79% (1/0)

O

N3 O

O H O

O

16

O

HO

OMe OBn

O

S Ph

H

OpMP

OB n

13

N3 O

O

5

O

O

OBn 12

2

BnO OMe

Ph

O

HO

4

O

O BnO Bn O 11

SPh

O

AcO AcO AcO

BnO O Me

N3 O

O O BnO

Ph

3

O

O BnO B nO 10 Ph

9

3

N3 O

AcO BnO

OMe

N3 O

O

disaccharide AcO

OH

N3 O

5

Ph

activator yield (α/β)a,b

acceptor

15

O

O

Ph

6

12

2

Ph2SO/Tf2O 64% (1/0)

AcO AcO AcO

N3 O O O O

O 17

7

MeOO C N3 O A cO BnO 8

9

Ph2SO/Tf2O 69% (0/1)

MeO OC N 3 O AcO B nO

O

O

SPh

OpMP

OB n

BnO

O Me OBn

O Bn

18

Ph2SO/Tf2O no rxn 8

a

12

NIS/TMSOTf 53% (1/2)

isolated yields. b anomeric ratios were determined by 1H NMR spectroscopy.

86   

86

8

Ph O O

MeOO C N3 O A cO BnO O

19

O OB n

OpMP

Deactivated 1‐Thiomannosazidopyranosides   

8, bearing an electron withdrawing ester moiety at the C5 position was investigated. Coupling of donor 8 with primary alcohol 9 gave exclusively β-linked disaccharide 18 in 69% yield. An attempt to glycosylate the secondary alcohol acceptor 12 using the same Ph2SO/Tf2O activation protocol did not result in satisfactory product formation. Monitoring the reaction by TLC analysis showed that the initial pre-activation of donor 8 with Ph2SO/Tf2O proceeded uneventfully. Addition of the acceptor and warming of the reaction mixture showed partial regeneration of the donor 8 in addition to the formation of several by-products. This observation indicates that under Ph2SO/Tf2O activation conditions the donor glycoside is not always fully converted into the intermediate α-triflate.31 Changing the activation system to NIS/TMSOTf21,32 led to a productive coupling of donor 8 with acceptor 12 affording disaccharide 19 in 53% yield as an anomeric mixture (α/β = 1/2). Finally, the assembly of fully protected trisaccharide 24 was investigated (Scheme 2). In its unprotected form, this trisaccharide [→3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-αD-GlcpNAc-(1→], is the repeating unit of a polysaccharide that constitutes 70% or more of the enterobacterial common antigen (ECA).33,34 The route of synthesis to trimer 24 was investigated starting from the non-reducing end D-fucose residue and using both a chemoselective27a and an orthogonal glycosylation strategy.35 In the chemoselective approach, 4-azido 1-thiofucose donor 2036 was coupled with 1-thiomannosaziduronic acid ester 7 to afford α-linked thiodisaccharide 22 in a yield of 55%.37 The result of this coupling was compared with an orthogonal glycosylation strategy in which 1-hydroxyl donor 21 was condensed with acceptor 7. To this end, fucose donor 21 was prepared via hydrolysis of the thiophenyl function in compound 20 using the NIS/TFA reagent system.38 Activation of donor 21 with 2.8 equiv. Ph2SO and 1.3 equiv. Tf2O for 1h at -40°C followed by the addition Scheme 2. N3 N3 O BnO BnO a

O

MeOOC N3 O HO BnO

+ R

BnO

b or c

BnO SPh

22: R = β-SPh 23: R = α/β-OH

9

OBn O

O BnO

O

MeOOC N3 O BnO SPh

24

N3 BnO

O

MeOOC N3 O BnO

O

OBn O

O d

HO

25

O

N3

N3

26

Reagents and conditions: (a) NIS, TFA, DCM/H2O (10/1), 72% (b) 20, Ph2SO, Tf2O, TTBP, DCM, -60°C then 7, 55% (c) 21, Ph2SO, Tf2O, TTBP, DCM, -60°C then 7, 67% (d) 22, 23, NIS, TMSOTf, DCM, -40°C, 65% (α/β = 1/2).

87   

87

Chapter 5   

of acceptor 7 gave α-linked thiodisaccharide 22 in 67% yield. Treatment of a mixture of 1thiodisaccharide donor 22 with 1,6-anhydro acceptor 2339 with the NIS/TMSOTf reagent combination finally afforded trisaccharide 24 in 65% yield as a separable anomeric mixture of α/β = 1/2.

Conclusion This Chapter describes the glycosylation properties of 1-thiomannosazidopyranosides bearing electron-withdrawing substituents on different positions of the carbohydrate core. It was found that 4,6-di-O-acetyl protection on 1-thiomannosazides sufficiently influences the electronic environment around the anomeric centre leading to reasonable β-selectivities. Acyl protection at the C3 position always gave exclusive α-glycoside formation. Furthermore, the uronic acid ester functionalized mannosazides (8 and 22) also showed good to excellent βselectivities.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Brüker DMX-400 and a Brüker AV-400 (400/100 MHz) and a Brüker AV500 (500/125 MHz) spectrometer. Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and Q-Star Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide and TTBP were removed by co-evaporation with toluene. TTBP was synthesized as described by Crich et al.25 Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography and TLC were of technical grade and distilled before use. Flash chromatography was performed on Fluka silica gel 60 (0.04 – 0.063 mm). TLC-analysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in water followed by charring at ~150°C. General Procedure for Glycosidations using Ph2SO/Tf2O: A solution of 1-thio mannopyranoside uronic acid ester (1 equiv.), diphenyl sulfoxide (1.3 equiv.) and tri-tert-butylpyrimidine (2.5 equiv.) in DCM (0.05M) was stirred over activated MS3Å for 30min. The mixture was cooled to -60°C before triflic acid anhydride (1.3 equiv.) was added. The mixture was allowed to warm to -50°C in 15min followed by addition of acceptor (1.5 equiv.) in DCM (0.15M). Stirring was continued and the reaction mixture was allowed to warm to rT. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with

88   

88

Deactivated 1‐Thiomannosazidopyranosides    EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the corresponding disaccharides. AcO

N3 O

Phenyl 4,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxy-1-thio-α-D-mannopyranoside (5): Known

compound 222 (381 mg, 0.9 mmol) was dissolved in 3 mL MeOH and a catalytic amount of SPh KOtBu was added. After TLC-analysis indicated complete reaction, the reaction mixture was neutralized using Amberlite IR120 H+-form. The reaction mixture was filtered and concentrated under reduced pressure. The crude oil was suspended in 3 mL acetonitrile followed by addition of 0.15 mL benzaldehyde dimethylacetal (1 mmol, 1.1 equiv.) and a catalytic amount of TsOH. After TLC-analysis indicated complete AcO BnO

reaction, 1 mL triethylamine was added and the reaction mixture was concentrated under reduced pressure. The crude oil is dissolved in 3 mL DMF and cooled to 0°C. Benzylbromide (0.14 mL, 1.2 mmol, 1.3 equiv.) and 48 mg sodium hydride (1.2 mmol, 1.3 equiv.) were added. After TLC-analysis indicated complete conversion, the reaction mixture was dissolved in 50 mL Et2O and washed with water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude oil was suspended in 10 mL MeOH and a catalytic amount of TsOH was added. The reaction mixture was refluxed for 1h followed by the addition of 1 mL triethylamine and concentration under reduced pressure. The crude oil was treated with a mixture of 5 mL pyridine and 2 mL acetic anhydride. After TLC-analysis indicated complete reaction, the reaction mixture was quenched by the addition of 1 mL MeOH, taken up in 50 mL Et2O and washed with water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using EtOAc/petroleum ether afforded 391 mg of the title compound 5 (0.79 mmol, 88% over the 5 steps) as a colorless oil. TLC: 50% EtOAc/PE; [α]D22: +12° (c = 0.8, CHCl3); IR (neat, cm-1): 740, 1001, 1118, 1218, 1373, 2102; 1H NMR (400 MHz, CDCl3) δ = 2.02 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac), 3.95 (dd, 1H, J = 9.2 Hz, J = 3.6 Hz, H-3), 4.05 – 4.09 (m, 2H, J = 2.4 Hz, H-2, H-6), 4.20 (dd, 1H, J = 12.4 Hz, J = 6.0 Hz, H-6), 4.33 – 4.38 (m, 1H, J = 6.0 Hz, J = 2.4 Hz, H-5), 4.62 (d, 1H, J = 12.0 Hz, CHHPh), 4.70 (d, 1H, J = 12.0 Hz, CHHPh), 5.31 (t, 1H, J = 9.6 Hz, H-4), 5.48 (d, 1H, J = 1.6 Hz, H-1), 7.26 – 7.55 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 20.8 (CH3 Ac), 62.2 (C-2), 62.4 (C-6), 67.6 (C-4), 69.9 (C-5), 72.6 (CH2 Bn), 76.7 (C-3), 85.9 (C-1), 127.9 – 131.8 (CH Arom), 132.7 (Cq SPh), 137.1 (Cq Bn), 169.5 (C=O Ac), 170.7 (C=O Ac); HRMS: [M+Na]+ calcd for C23H25O6N3SNa 494.13563, found 494.13583. Phenyl 2-azido-3-O-benzyl-2-deoxy-1-thio-α-D-mannopyranosiduronic acid (6): To a vigorously stirred solution of 505 mg compound 4 (1.3 mmol) in 10 mL DCM and 2 mL H2O SPh were added 41 mg TEMPO (0.26 mmol, 0.2 equiv.) and 966 mg BAIB (3 mmol, 2.5 equiv.). Stirring was allowed until TLC indicated complete conversion of the starting material into a lower running HOOC N3 O

HO BnO

product. The reaction mixture was quenched by the addition of 25 mL Na2S2O3 solution (10% in H2O). The mixture was then extracted twice with 10 mL EtOAc and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash column chromatography (EtOAc/PE, 2% (v/v) AcOH) afforded 340 mg of the title compound 6 (0.85 mmol, 65%) as a colorless oil. TLC: 50% EtOAc/PE (5% AcOH); 1H NMR (600 MHz, CDCl3) δ = 3.90 (dd, 1H, J = 8.4 Hz, J = 3.4 Hz, H-3), 3.99 (s, 1H, H-2), 4.25 (t, 1H, J = 8.6 Hz, H-4), 4.64 (d, 1H, J = 8.4 Hz, H-5), 4.74 (d, 1H, J = 11.7 Hz, CHHPh), 4.84 (d, 1H, J = 11.7 Hz, CHHPh), 5.51 (d, 1H, J = 3.9 Hz, H-1), 7.26 – 7.59 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 61.1 (C-2), 68.5 (C-4), 71.8 (C-5), 73.4 (CH2 Bn), 77.9 (C-3), 85.9 (C-1), 127.9 – 132.2 (CH Arom), 132.1 (Cq SPh), 137.2 (Cq Bn), 172.57 (C=O COOH); 13C-GATED (100 MHz, CDCl3): 85.9 (J = 169 Hz, C-1); HRMS: [M+Na]+ calcd for C19H19O5N3SNa 424.09376, found 424.09389.

89   

89

Chapter 5    Me OOC N 3 O

Methyl (phenyl 2-azido-3-O-benzyl-2-deoxy-1-thio-α-D-mannopyranoside) uronate (7): To a stirred solution of 340 mg compound 6 (0.85 mmol) in 5 mL DMF was added 60 µL MeI (1 SP h mmol, 1.2 equiv.) and 200 mg K2CO3. The solution was stirred overnight, taken up in EtOAc and washed with H2O. The aqueous phase was extracted with EtOAc. The combined organic phase was dried HO B nO

(MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography (EtOAc/PE) afforded 245 mg of the title compound 7 (0.6 mmol, 71%) as a colorless oil. TLC: 50% EtOAc/PE; [α]D22: +69° (c = 1.4, CHCl3); IR (neat, cm-1): 740, 1001, 1118, 1218, 1373, 1747, 2102; 1H NMR (400 MHz, CDCl3) δ = 3.04 (bs, 1H, OH-4), 3.71 (s, 1H, CH3 COOMe), 3.87 (dd, 1H, J = 8.0 Hz, J = 3.4 Hz, H-3), 3.95 (t, 1H, J = 3.4 Hz, H-2), 4.31 (dq, J = 7.9 Hz, J = 2.1 Hz, H-4), 4,60 (d, 1H, J = 7.9 Hz, H-5), 4.72 (d, 1H, J = 11.6 Hz, CHHPh), 4.78 (d, 1H, J = 11.6 Hz, CHHPh), 5.52 (d, 1H, J = 1.4 Hz, H-1), 7.24 – 7.51 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.6 (CH3 COOMe), 61.0 (C-2), 68.3 (C-4), 72.9 (C-5), 73.2 (CH2 Bn), 77.9 (C-3), 85.4 (C-1), 127.9 – 131.9 (CH Arom), 137.1 (Cq Bn), 169.8 (C=O COOMe); HRMS: [M+NH4]+ calcd for C20H25O5N4S 433.15457, found 433.15418. Methyl (phenyl 4-acetyl-2-azido-3-O-benzyl-2-deoxy-1-thio-α-D-mannopyranoside) uronate (8): Compound 7 (245 mg, 0.6 mmol) was treated with 3 mL pyridine/Ac2O (3:1) solution for 8h followed by the addition of 2 mL MeOH. The reaction mixture was taken up in EtOAc and SPh washed with 2M HCl and saturated NaHCO3. The organic phase is dried (MgSO4), filtered and concentrated MeOOC N3 O

AcO BnO

under reduced pressure. Flash column chromatography (EtOAc/PE) afforded the title compound 8 (274 mg, 0.6 mmol, quant.) as a colorless oil. TLC: 50% EtOAc/PE (5% AcOH); [α]D22: +37° (c = 1, CHCl3); IR (neat, cm-1): 740, 1001, 1118, 1218, 1373, 1747, 2102; 1H NMR (500 MHz, CDCl3) δ = 2.05 (s, 3H, CH3 Ac), 3.85 (bs, 4H, H-2, CH3 COOMe), 3.95 (t, 1H, J = 3.5 Hz, H-3), 4.56 (d, 1H, J = 2.5, H-5), 4.62 (d, 1H, J = 11.5 Hz, CHHPh), 4.65 (d, 1H, J = 11.5 Hz, CHHPh), 5.54 (t, 1H, J = 3.5 Hz, J = 2.5 Hz, H-4), 5.72 (d, 1H, J = 9.0 Hz, H-1), 7.25 – 7.66 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.2 (CH3 COOMe), 57.7 (C-2), 68.2 (C-4), 72.9 (CH2 Bn), 73.2 (C-5), 74.8 (C-3), 80.7 (C-1), 127.8 – 132.2 (CH Arom), 131.7 (Cq SPh), 136.3 (Cq Bn), 167.9 (C=O COOMe or Ac), 169.5 (C=O Ac or COOMe); HRMS: [M+H]+ calcd for C22H24O6N3S 458.13803, found 458.13934. Methyl (2,3,4-tri-O-benzyl-6-O-(4,6-di-O-acetyl-2-azido-3-O-benzyl-2-deoxyα/β-D-mannopyranosyl)-α-D-glucopyranoside (10): Disaccharide 10 was O O obtained from donor 5 and acceptor 9 according to the general procedure for BnO BnO OMe BnO glycosidations using the Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE; αanomer: 1H NMR (500 MHz, CDCl3) δ = 2.00 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac), 3.33 (s, 3H, CH3 AcO AcO BnO

N3 O

COOMe), 3.39 (t, 1H, J = 9.0 Hz, H-4), 3.49 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz, H-2), 3.61 (d, 1H, J = 11.5 Hz, H6), 3.67 – 3.74 (m, 2H, H-5, H-5’), 3.78 (dd, 1H, J = 11.5 Hz, J = 4.5 Hz, H-6), 3.86 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-3’), 3.89 (d, 1H, J = 1.5 Hz, H-2’), 3.95 (dd, 1H, J = 12.0 Hz, J = 2.5 Hz, H-6’), 3.99 (t, 1H, J = 9.0 Hz, H-3), 4.03 (dd, 1H, J = 12.0 Hz, J = 4.5 Hz, H-6’), 4.45 (d, 1H, J = 11.0 Hz, CHHPh), 4.53 (d, 1H, J = 12.0 Hz, CHHPh), 4.61 (d, 1H, J = 3.5 Hz, H-1), 4.64 (d, 1H, J = 12.0 Hz, CHHPh), 4.67 (d, 1H, J = 11.5 Hz, CHHPh), 4.80 (d, 2H, J = 11.0 Hz, CHHPh, CHHPh), 4.85 (s, 1H, H-1’), 4.92 (d, 1H, J = 11.5 Hz, CHHPh), 5.02 (d, 1H, J = 11.0 Hz, CHHPh), 5.23 (t, 1H, J = 9.5 Hz, H-4’), 7.21 – 7.40 (m, 20H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 20.7 (CH3 Ac), 20.8 (CH3 Ac), 55.2 (CH3 OMe), 60.6 (C-2’), 62.1 (C-6’), 66.8 (C-6), 67.2 (C-4’), 68.9 (C5’), 68.6 (C-5), 71.9 (CH2 Bn), 73.2 (CH2 Bn), 74.9 (CH2 Bn), 75.7 (C-3’), 75.8 (CH2 Bn), 77.6 (C-4), 79.9 (C2), 82.0 (C-3), 97.9 (C-1), 98.4 (C-1’), 127.3 – 128.6 (CH Arom), 137.2 (Cq Bn), 137.9 (Cq Bn), 138.1 (Cq Bn), 138.6 (Cq Bn), 169.4 (C=O Ac), 170.7 (C=O Ac); 13C-GATED (125 MHz, CDCl3): 97.9 (JC1,H1 = 171 Hz, C-1), 98.4 (JC1’,H1’ = 175 Hz, C-1’); HRMS: [M+H]+ calcd for C45H52O12N3 826.35455, found 826.35550. β-anomer:

90   

90

Deactivated 1‐Thiomannosazidopyranosides    22

-1

[α]D : -17° (c = 0.8, CHCl3); IR (neat, cm ): 698, 738, 754, 1004, 1039, 1068, 1132, 1240, 1367, 1743, 2104; H NMR (400 MHz, CDCl3) δ = 2.01 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac), 3.34 (s, 3H, CH3 OMe), 3.36 (t, 1H, J = 9.2 Hz, H-4), 3.38 – 3.41 (m, 1H, H-5’), 3.46 (dd, 1H, J = 9.6 Hz, J = 4.5 Hz, H-3), 3.49 (d, 1H, J = 8.0 Hz, H-2), 3.51 (m, 1H, J = 3.6 Hz, H-6), 3.75 (d, 1H, J = 4.5 Hz, H-2), 4.00 (t, 1H, J = 9.2 Hz, H-3), 4.07 – 4.11

1

(m, 2H, H-6, H-6’), 4.17 (dd, 1H, J = 12.0 Hz, J = 5.2 Hz, H-6’), 4.26 (s, 1H, H-1’), 4.53 (d, 1H, J = 11.6 Hz, CHHPh), 4.55 (d, 1H, J = 12.4 Hz, CHHPh), 4.56 (s, 1H, H-1), 4.62 (d, 1H, J = 12.0 Hz, CHHPh), 4.67 (d, 1H, J = 12.4 Hz, CHHPh), 4.77 (d, 1H, J = 12.0 Hz, CHHPh), 4.80 (d, 1H, J = 10.8 Hz, CHHPh), 4.87 (d, 1H, J = 11.6 Hz, CHHPh), 5.00 (d, 1H, J = 10.8 Hz, CHHPh), 5.15 (t, 1H, J = 9.6 Hz, H-4’), 7.23 – 7.36 (m, 20H, H Arom); 13 C NMR (100 MHz, CDCl3) δ = 20.7 (CH3 Ac), 20.7 (CH3 Ac), 55.1 (CH3 OMe), 61.3 (C-2’), 62.6 (C-6’), 67.3 (C-4’), 68.7 (C-6), 69.5 (C-5), 71.8 (CH2 Bn), 72.4 (C-5’), 73.4 (CH2 Bn), 74.6 (CH2 Bn), 75.7 (CH2 Bn), 77.2 (C-3’), 77.6 (C-4), 79.8 (C-2), 81.9 (C-3), 97.8 (C-1), 99.8 (C-1’), 127.3 – 128.6 (CH Arom), 137.2 (Cq Bn), 138.0 (Cq Bn), 138.2 (Cq Bn), 138.6 (Cq Bn), 169.3 (C=O Ac), 170.8 (C=O Ac); 13C-GATED (100 MHz, CDCl3): 97.8 (JC1,H1 = 171 Hz, C-1), 99.8 (JC1’,H1’ = 158 Hz, C-1’); HRMS: [M+Na]+ calcd for C45H51O12N3Na 848.33650, found 848.33642. para-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-3-O-(4,6-di-O-acetyl-2azido-3-O-benzyl-2-deoxy-α/β-D-mannopyranosyl)-β-D-galactopyranoside (13): Disaccharide 13 was obtained from donor 5 and acceptor 12 according to

Ph AcO AcO BnO

O O

N3 O

O

the general procedure for glycosidations using the Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE; β-anomer: IR (neat, cm-1): 696, 1028, 1035, 1217, 1506, 1733, 2102; 1H NMR (400 MHz, CDCl3) δ = 1.98 (s, 3H, CH3 Ac), 2.05 (s, 3H, CH3 Ac), 3.27 (dd, 1H, J = 9.5 Hz, J = 3.6 Hz, H-3’), 3.51 (s, 1H, H-5), 3.68 (d, 1H, J = 9.8 Hz, H-5’), 3.71 (s, 3H, CH3 OMe pMP), 3.76 (bs, 1H, H-2’), 3.89 (dd, 1H, J = 10.0 Hz, J = 3.4 Hz, H-3), 4.07 (dd, 1H, J = 12.5 Hz, J = 1.2 Hz, H-6), O

OpMP

OBn

4.17 (dd, 1H, J = 10.0 Hz, J = 7.8 Hz, H-2), 4.20 (d, 1H, J = 13.2 Hz, H-6), 4.36 (s, 1H, H-4), 4.43 (d, 1H, J = 12.2 Hz, CHHPh), 4.56 (d, 1H, J = 12.2 Hz, CHHPh), 4.66 (d, 1H, J = 11.7 Hz, CHHPh), 4.88 (s, 1H, H-1’), 4.90 (d, 1H, J = 7.8 Hz, H-1), 5.07 (d, 1H, J = 11.7 Hz, CHHPh), 5.23 (t, 1H, J = 9.6 Hz, H-4’), 5.59 (s, 1H, CHPh), 6.81 (d, 1H, J = 9.1 Hz, 2xCH pMP), 7.05 (d, 1H, J = 9.1 Hz, 2xCH pMP), 7.25 – 7.59 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 20.9 (CH3 Ac), 55.6 (CH3 OMe pMP), 60.9 (C-2’), 66.7 (C-5), 68.1 (C-4), 68.8 (C-6), 72.2 (CH2 Bn), 72.9 (C-5’), 75.3 (CH2 Bn), 75.6 (C-4), 77.1 (C-3), 77.3 (C3’), 78.9 (C-2), 100.4 (C-1’), 100.7 (CHPh), 103.2 (C-1), 114.4 (CH Arom pMP), 118.8 (CH Arom pMP), 126.4 – 128.9 (CH Arom), 137.2 (Cq Bn), 137.7 (Cq Bn), 151.3 (Cq pMP), 155.4 (Cq pMP), 167.4 (C=O Ac or COOMe), 169.3 (C=O Ac or COOMe); 13C-GATED (100 MHz, CDCl3): 100.4 (JC1’,H1’ = 161 Hz, C-1’), 103.2 (JC1,H1 = 163 Hz, C-1); HRMS: [M+NH4]+ calcd for C43H49O13N4 829.32961, found 829.32829.

AcO AcO AcO

N3 O O

OMe OBn

O

Methyl (2,3,4-tri-O-benzyl-6-O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranosyl)-α-D-glucopyranoside (14): Disaccharide 14 was obtained from donor 2 and acceptor 9 according to the general procedure for glycosidations using the

Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE; IR (neat, cm-1): 698, 734, 819, 999, 1026, 1101, 1132, 1217, 1506, 1747, 2106; 1H NMR (400 MHz, CDCl3) δ = 2.06 (s, 3H, CH3 Ac), 2.07 (s, 3H, CH3 Ac), 2.13 (s, 3H, CH3 Ac), 3.83 (s, 3H, CH3 OMe), 3.45 (t, 1H, J = 9.2 Hz, H-4), 3.53 (dd, 1H, J = 9.6 Hz, J = 3.6 Hz, H-3), 3.64 (d, 1H, J = 9.6 Hz, H-6), 3.76 - 3.80 (m, 1H, H-5), OBn BnO

3.81 (dd, 1H, J = 9.6 Hz, J = 4.0 Hz, H-6), 3,82 – 3.86 (m, 1H, H-5'), 3.97 – 4.03 (m, 3H, H-3, H-2' and H-6'), 4.09 (dd, 1H, J = 12.0 Hz, J = 4.8 Hz, H-6'), 4.58 (d, 1H, J = 3.6 Hz, H-1), 4 59 (d, 1H, J = 11.2 Hz, CHHPh), 4.68 (d, 1H, J = 12.0 Hz, CHHPh), 4.78 (d, 1H, J = 12.0 Hz, CHHPh), 4.80 (d, 1H, J = 10.8 Hz, CHHPh), 4.88

91   

91

Chapter 5    (d, 1H, J = 1.6 Hz, H-1'), 4.97 (d, 1H, J = 11.2 Hz, CHHPh), 4.99 (d, 1H, J = 10.8 Hz, CHHPh), 5.25 (t, 1H, J = 10.0 Hz, H-4'), 5.31 (t, 1H, J = 10.0 Hz, H-3'), 7.30 – 7.43 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.5 (CH3 Ac), 20.6 (CH3 Ac), 20.7 (CH3 Ac), 55.2 (CH3 OMe), 60.5 (C-2'), 61.8 (C-6'), 65.8 (C-4'), 66.5 (C-6), 68.5 (C-5'), 69.9 (C-5), 70.9 (C-3'), 73.3 (CH2 Bn), 75.2 (CH2 Bn), 75.8 (CH2 Bn), 80.0 (C-4), 81.1 (C-2), 82.1 (C-3), 97.9 (C-1), 98.2 (C-1'), 127.5 – 128.5 (CH Arom), 138.0 (Cq Bn), 138.1 (Cq Bn), 138.5 (Cq Bn), 169.5 (C=O Ac), 169.8 (C=O Ac), 170.6 (C=O Ac); 13C-GATED (100 MHz, CDCl3): 97.9 (JC1,H1 = 164 Hz, C-1), 98.2 (JC1’,H1’ = 174 Hz, C-1’); HRMS: [M+NH4]+ calcd for C40H51O13N4 795.34471, found 795.34144.

AcO AcO AcO

3-O-(3,4,6-Tri-O-acetyl-2-azido-2-deoxy-α-D-mannopyranosyl)-1,2;5,6-di-O-isopropyl-

O

N3 O

O H O

O O

O

idene-α-D-glucofuranose (16): Disaccharide 16 was obtained from donor 2 and acceptor 15 according to the general procedure for glycosidations using the Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE; IR (neat, cm-1): 698, 734, 819, 999, 1026, 1101, 1132, 1217, 1506, 1747, 2106; 1H NMR (400 MHz, CDCl3) δ = 1.27 (s, 3H, CH3 isoprop), 1.32 (s, 3H, CH3 isoprop), 1.37 (s, 3H, CH3 isoprop), 1.41 (s, 3H, CH3 isoprop), 2.06 (s,

3H, CH3 Ac), 2.10 (s, 3H, CH3 Ac), 2.12 (s, 3H, CH3 Ac), 3.97 – 4.05 (m, 2H, H-5', H6'), 4.06 (dd, 1H, J = 3.0 Hz, J = 1.0 Hz, H-2'), 4.07 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, H-4), 4.12 – 4.17 (m, 3H, H-6, H-5' and H-6'), 4.25 (dd, 1H, J = 12.0 Hz, J = 5.5 Hz, H-6), 4.28 (d, 1H, J = 3.0 Hz, H-3), 4.57 (d, 1H, J = 3.5 Hz, H-2), 5.19 (d, 1H, J = 1.0 Hz, H-1'), 5.28 (t, 1H, J = 9.5 Hz, H-4'), 5.23 (dd, 1H, J = 10.0 Hz, J - 4.0 Hz, H-3'), 5.91 (d, 1H, J = 3.5 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ = 20.5 (CH3 Ac), 20.6 (CH3 Ac), 20.7 (CH3 Ac), 25.2 (CH3 isoprop), 26.2 (CH3 isoprop), 26.8 (2xCH3 isoprop), 61.2 (C-2'), 62.3 (C-6), 66.0 (C-3'), 67.9 (C6'), 69.3 (C-5'), 70.4 (C-4'), 72.5 (C-5), 81.3 (C-4), 81.7 (C-3), 83.9 (C-2), 99.1 (C-1'), 105.3 (C-1), 109.5 (Cq isoprop), 112.1 (Cq isoprop), 169.5 (C=O Ac), 169.9 (C=O Ac), 170.7 (C=O Ac); 13C-GATED (100 MHz, CDCl3): 99.1 (JC1',H1' = 171 Hz, C-1'), 105.3 (JC1,H1 = 181 Hz, C-1’); HRMS: [M+Na]+ calcd for C24H35O13N3Na 596.20621, found 596.20586. para-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-3-O-(3,4,6-tri-O-acetyl-2azido-2-deoxy-α-D-mannopyranosyl)-β-D-galactopyranoside (17): Disaccharide 17 was obtained from donor 2 and acceptor 12 according to the general procedure

Ph AcO AcO AcO

N3 O O O O

for glycosidations using the Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE; IR (neat, cm-1): 698, 734, 819, 999, 1026, 1101, 1132, 1217, 1506, 1747, 2106; 1 H NMR (400 MHz, CDCl3) δ = 1.97 (s, 3H, CH3 Ac), 2.03 (s, 3H, CH3 Ac), 2.09 (s, 3H, CH3 Ac), 3.49 (s, 1H, H-5), 3.78 (s, 3H, CH3 OMe pMP), 3.86 (dd, 1H, J = 10.0 Hz, J = 3.6 Hz, H-3), 3.93 (d, 1H, J = 3.2 Hz, H-6'), 4.03 – 4.06 (m, 2H, H-2, H-2'), 4.08 (dt, 1H, J = 11.6 Hz, J = 1.6 Hz, H-6), 4.14 (dq, 1H, J = 10.4 Hz, J = 2.8 O

OpMP

OBn

Hz, H-5'), 4.32 (s, 1H, J = 3.2 Hz, H-4), 4.37 (dd, 1H, J = 12.4 Hz, J = 1.2 Hz, H-6), 4.78 (d, 1H, J = 10.4 Hz, CHHPh), 4.90 (d, 1H, J = 8.0 Hz, H-1), 5.05 (d, 1H, J = 1.2 Hz, H-1'), 5.06 (d, 1H, J = 10.4 Hz, CHHPh), 5.32 (t, 1H, J = 10.0 Hz, H-4'), 5.47 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz, H-3'), 5.56 (s, 1H, CHPh), 6.82 (d, 1H, J = 9.1 Hz, CH Arom pMP), 7.05 (d, 1H, J = 9.1 Hz, CH Arom pMP), 7.25 – 7.59 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.5 (CH3 Ac), 20.7 (CH3 Ac), 20.7 (CH Ac), 55.6 (CH3 pMP), 61.5 (C-2'), 61.6 (CH2 Bn), 65.6 (C-4'), 66.3 (C-5), 68.5 (C-5'), 69.2 (C-6), 71.0 (C-3'), 71.1 (C-4), 74.3 (C-3), 76.3 (C-2), 93.8 (C-1'), 101.1 (CHPh), 103.3 (C-1), 114.5 (CH Arom pMP), 118.9 (CH Arom pMP), 126.4 - 129.1 (CH Arom), 137.5 (Cq Bn), 138.0 (Cq Bn), 151.4 (Cq pMP), 155.5 (Cq pMP), 169.4 (C=O Ac), 169.7 (C=O Ac), 170.6 (C=O Ac); 13CGATED (100 MHz, CDCl3): 93.8 (JC1’,H1’ = 172 Hz, C-1’), 103.3 (JC1,H1 = 158 Hz, C-1); HRMS: [M+NH4]+ calcd for C39H47O14N4 795.30833, found 795.30491.

92   

92

Deactivated 1‐Thiomannosazidopyranosides    MeOOC N3 O AcO BnO

OMe OBn

O

O

BnO

OBn

Methyl (2,3,4-tri-O-benzyl-6-O-(methyl 4-O-acetyl-2-azido-3-O-benzyl-2deoxy-β-D-mannopyranosyluronate)-α-D-glucopyranoside (18): Disaccharide 18 was obtained from donor 8 and acceptor 9 according to the general procedure for glycosidations using the Ph2SO/Tf2O activator system. TLC: 30% EtOAc/PE;

22

[α]D : -16° (c = 1, CHCl3); IR (neat, cm-1): 698, 736, 754, 1033, 1070, 1105, 1180, 1224, 2113; 1H NMR (500 MHz, CDCl3) δ = 2.04 (s, 3H, CH3 Ac), 3.31 (s, 3H, CH3 OMe), 3.35 (t, 1H, J = 9.5 Hz, H-4), 3.44 (dd, 1H, J = 10.5 Hz, J = 6.5 Hz, H-6), 3.48 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-2), 3.55 (dd, 1H, J = 9.0 Hz, J = 3.5 Hz, H3’), 3.69 (s, 4H, H-2 and CH3 COOMe), 3.73 (d, 1H, J = 9.5 Hz, H-5’), 3.79 (m, 1H, J = 8.0 Hz, J = 6.5 Hz, J = 1.5 Hz, H-5), 4.00 (t, 1H, J = 9.5 Hz, H-3), 4.10 (dd, 1H, J = 8.0 Hz, J = 1.5 Hz, H-6), 4.28 (s, 1H, H-1’), 4.53 (d, 1H, J = 11.0 Hz, CHHPh), 4.54 (s, 1H, H-1), 4.61 (d, 1H, J = 11.0 Hz, CHHPh), 4.62 (d, 1H, J = 11.0 Hz, CHHPh), 4.66 (d, 1H, J = 11.0 Hz, CHHPh), 4.76 (d, 1H, J = 11.0 Hz, CHHPh), 4.79 (d, 1H, J = 11.0 Hz, CHHPh), 4.86 (d, 1H, J = 11.0 Hz, CHHPh), 4.98 (d, 1H, J = 11.0 Hz, CHHPh), 5.31 (t, 1H, J = 9.0 Hz, H-4’), 7.23 – 7.37 (m, 20H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 20.7 (CH3 Ac), 52.7 (CH3 COOMe), 55.1 (CH3 OMe), 68.2 (C-4’), 68.8 (C-6), 69.6 (C-5), 72.2 (CH2 Bn), 73.2 (C-5’), 73.4 (CH2 Bn), 74.6 (CH2 Bn), 75.7 (CH2 Bn), 75.7 (C-3’), 77.6 (C-4), 79.8 (C-2), 81.9 (C-3), 97.6 (C-1), 99.7 (C-1’), 127.6 – 128.6 (CH Arom), 137.1 (Cq Bn), 138.0 (Cq Bn), 138.3 (Cq Bn), 138.4 (Cq Bn), 138.6 (Cq Bn), 167.5 (C=O Ac or COOMe), 169.3 (C=O Ac or COOMe); 13C-GATED (125 MHz, CDCl3): 97.8 (JC1,H1 = 170 Hz, C-1), 99.7 (JC1,H1 = 160 Hz, C-1’); HRMS: [M+Na]+ calcd for C41H49O12N3Na 834.32084, found 834.32100. Ph MeOOC N3 O AcO BnO

O O

O O

OpMP

para-Methoxyphenyl (2-O-benzyl-4,6-O-benzylidene-3-O-(methyl 4-O-acetyl-2azido-3-O-benzyl-2-deoxy-α/β-D-mannopyranosyluronate)-β-D-galactopyranoside (19): A solution of 41 mg donor 8 (0.09 mmol) and 70 mg acceptor 12 (0.15 mmol, 1.5 equiv.) in 3 mL DCM was stirred over activated MS3Å for 30min

OBn

before 29 mg N-iodosuccinimide (0.13 mmol, 1.3 equiv.) was added. The mixture was cooled to -40°C and 5 µL TMSOTf (cat.) was added. Stirring was continued and the reaction mixture was allowed to warm to rT. When TLC-analysis indicated complete conversion, 0.2 mL triethylamine was added. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded 40 mg of the title compound 19 (0.048 mmol, 53%, (α/β=1/2) as a colorless oil. TLC: 30% EtOAc/PE; IR (neat, cm-1): 696, 734, 746, 819, 1045, 1083, 1220, 1365, 1506, 1743, 2108; β-anomer: 1H NMR (400 MHz, CDCl3) δ = 2.04 (s, 3H, CH3 Ac), 3.28 (dd, 1H, J = 9.5 Hz, J = 3.6 Hz, H-3’), 3.51 (bs, 1H, H-5), 3.65 (d, 1H, J = 9.8 Hz, H-5’), 3.71 (s, 3H, CH3 pMP or COOMe), 3.75 (d, 1H, J = 3.6 Hz, H-2’), 3.77 (s, 3H, CH3 COOMe or pMP), 3.89 (dd, 1H, J = 10.0 Hz, J = 3.4 Hz, H-3), 4.03 – 4.08 (m, 1H, H-6), 4.18 (dd, 1H, J = 10.0 Hz, J = 7.8 Hz, H-2), 4.34 (d, 1H, J = 13.2 Hz, H-6), 4.37 (s, 1H, H-4), 4.45 (d, 1H, J = 12.2 Hz, CHHPh), 4.58 (d, 1H, J = 12.2 Hz, CHHPh), 4.66 (d, 1H, J = 11.7 Hz, CHHPh), 4.88 (s, 1H, H-1’), 4.90 (d, 1H, J = 7.8 Hz, H-1), 5.08 (d, 1H, J = 11.7 Hz, CHHPh), 5.21 (t, 1H, J = 9.6 Hz, H-4’), 5.60 (s, 1H, CHPh), 6.82 (d, 1H, J = 9.1 Hz, CH Arom pMP), 7.05 (d, 1H, J = 9.1 Hz, CH Arom pMP), 7.25 – 7.59 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 20.6 (CH3 Ac), 52.7 (CH3 pMP or COOMe), 55.6 (CH3 COOMe or pMP), 60.9 (C-2’), 66.7 (C-5), 68.2 (C-5), 68.8 (C-6), 72.1 (CH2 Bn), 73.2 (C5’), 75.3 (CH2 Bn), 75.6 (C-4), 77.1 (C-3), 77.3 (C-3’), 78.9 (C-2), 100.4 (C-1’), 100.7 (CHPh), 103.2 (C-1), 114.4 (CH Arom pMP), 118.7 (CH Arom pMP), 126.4 – 128.9 (CH Arom), 137.2 (Cq Bn), 137.7 (Cq Bn), 138.6 (Cq Bn), 151.4 (Cq pMP), 155.4 (Cq pMP), 167.5 (C=O COOMe or Ac), 169.3 (C=O Ac or COOMe); 13CGATED (100 MHz, CDCl3): 100.4 (JC1’,H1’ = 163 Hz, C-1’), 103.2 (JC1,H1 = 161 Hz, C-1); α-anomer: 13CGATED (100 MHz, CDCl3): 93.0 (JC1’,H1’ = 170 Hz, C-1’), 101.0 (JC1,H1 = 161 Hz, C-1); HRMS: [M+Na]+ calcd for C43H45O13N3Na 834.28446, found 834.28513.

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Chapter 5    4-Azido-2,3-di-O-benzyl-4-deoxy-α/β-D-fucopyranose (21): To a vigorously stirred solution of 119 mg compound 2040 (0.26 mmol) in 1 mL DCM/H2O (10/1 v/v) was added 58 mg NIS OH BnO (0.26 mmol, 1 equiv.) and 20 µL TFA (0.26 mmol, 1 equiv.). The mixture was stirred for 15min. before 10 mL Na2S2O3 (10% in H2O) and 10 mL NaHCO3 (sat. aq.) were added. The biphasic mixture N3

O

BnO

was diluted with 50 mL EtOAc and extracted. The aqueous phase was extracted twice with 25 mL EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using (EtOAc/PE) afforded 69 mg of the title compound 21 (0.19 mmol, 73%) as a colorless oil. TLC: 50% EtOAc/PE; IR (neat, cm-1): 609, 1041, 1091, 1222, 2098; α-anomer: 1H NMR (500 MHz, CDCl3) δ = 1.24 (d, 3H, J = 6.5 Hz, H-6), 3.26 (bs, 1H, OH-1), 3.74 (d, 1H, J = 3.5 Hz, H-4), 3.86 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-2), 4.03 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-3), 4.18 (q, 1H, J = 6.5 Hz, H-5), 4.70 (d, 1H, J = 11.5 Hz, CHHPh), 4.77 (d, 1H, J = 11.5 Hz, CHHPh), 4.80 (d, 1H, J = 11.5 Hz, CHHPh), 4.84 (d, 1H, J = 11.5 Hz, CHHPh), 5.18 (d, 1H, J = 3.5 Hz, H-1), 7.29 – 7.42 (m, 10H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 17.3 (C-6), 64.5 (C-4), 64.6 (C-5), 72.8 (CH2 Bn), 73.7 (CH2 Bn), 76.0 (C-2), 77.8 (C-3), 91.6 (C-1), 127.7 – 128.4 (CH Arom), 137.8 (Cq Bn), 137.9 (Cq Bn); β-anomer: 1H NMR (500 MHz, CDCl3) δ = 1.31 (d, 3H, J = 6.5 Hz, H-6), 3.55 (q, 1H, J = 6.5 Hz, H-5), 3.61 (t, 1H, J = 9.5 Hz, J = 8.5, H-2), 3.64 (d, 1H, J = 3.5 Hz, H-4), 3.68 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-3), 3.70 (bs, 1H, OH-1), 4.57 (bd, 1H, J = 8.5 Hz, H-1), 4.76 (d, 1H, J = 11.5 Hz, CHHPh), 4.82 (d, 1H, J = 11.5 Hz, CHHPh), 4.83 (d, 1H, J = 11.5 Hz, CHHPh), 4.91 (d, 1H, J = 11.5 Hz, CHHPh), 7.29 – 7.42 (m, 10H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 17.4 (C-6), 63.6 (C-4), 69.2 (C-5), 72.9 (CH2 Bn), 75.2 (CH2 Bn), 80.2 (C-2), 81.1 (C-3), 97.5 (C-1), 127.7 – 128.4 (CH Arom), 137.7 (Cq Bn), 138.3 (Cq Bn); HRMS: [M+Na]+ calcd for C20H23O4N3Na 392.15808, found 392.15813. Methyl (phenyl 2-azido-3-O-benzyl-2-deoxy-4-O-(4-azido-2,3-di-O-benzyl-4deoxy-α-D-fucopyranosyl)-α-D-mannopyranoside) uronate (22): Chemoselective

N3 O BnO

MeOOC N3 O BnO

glycosyl-ation: Disaccharide 22 was obtained from donor 20 and acceptor 7 according to the general procedure for glycosidations using the Ph2SO/Tf2O SPh activator system. Orthogonal glycosylation: A solution of 55 mg of donor 21 (0.15 mmol), 85 mg diphenylsulfoxide (0.42 mmol, 2.8 equiv.) and 112 mg tri-tert-butylpyrimidine (0.45 mmol, 3 equiv.) in 3 mL DCM was stirred over activated 100 mg MS3Å for 30min. The mixture was cooled to -60°C and BnO

O

33 µL Tf2O (0.20 mmol, 1.3 equiv.) was added. The mixture was allowed to warm to -40°C in 1h followed by addition of 78 mg acceptor 9 (0.17 mmol, 1.1 eq) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to 0°C. The reaction mixture was taken up in 25 mL EtOAc and washed with water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (10% EtOAc/PE) and removal of the eluent afforded 73 mg of the title compound 22 (0.095 mmol, 67%) as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: +3° (c = 0.3, CHCl3); IR (neat, cm-1): 609, 1041, 1091, 1222, 1710, 2098; 1H NMR (400 MHz, CDCl3) δ = 1.21 (d, 1H, J = 6.5 Hz, C-6’), 3.45 (dd, 1H, J = 10.0 Hz, J = 3.5 Hz, H-4), 3.47 (s, 3H, CH3 COOMe), 3.67 – 3.69 (m, 1H, H-3’, H-4’), 3.77 (dd, 1H, J = 9.5 Hz, J = 3.5 Hz, H-2’), 3.83 (q, 1H, J = 6.5 Hz, H-5’), 3.93 (t, 1H, J = 3.5 Hz, H-3), 4.23 (dd, 1H, J = 4.3 Hz, J = 2.0 Hz, H-2), 4.51 (d, 2H, J = 11.5 Hz, 2xCHHPh), 4.56 (d, 1H, J = 11.5 Hz, CHHPh), 4.61 (d, 1H, J = 2.0 Hz, H-1), 4.68 (d, 1H, J = 11.5 Hz, CHHPh), 4.75 (d, 1H, J = 11.5 Hz, CHHPh), 4.76 (d, 1H, J = 11.5 Hz, CHHPh), 4.77 (d, 1H, J = 3.5 Hz, H-1’), 5.62 (d, 1H, J = 10.0 Hz, H-5), 7.25 – 7.67 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 17.3 (C-6’), 52.1 (CH3 COOMe), 57.1 (C-4), 64.4 (C-3’or C-4’), 65.3 (C-5’), 72.9 (CH2 Bn), 73.1 (CH2 Bn), 73.8 (CH2 Bn), 75.0 (C-1), 75.3 (C-2), 75.9 (C-2’), 76.1 (C-3), 77.8 (C-4’ or C-3’), 79.6 (C-5), 99.7 (C-1’), 124.7 – 134.1 (CH Arom), 130.9 (Cq SPh), 136.6 (Cq Bn), 137.9 (Cq Bn), 138.2 (Cq Bn), 168.9 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 75.0 (JC1,H1 = could not be determined), 96.1 (JC1’,H1’ = 166 Hz, C1’); HRMS: [M+Na]+ calcd for C40H42O8N6SNa 789.26770, found 789.26819.

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Deactivated 1‐Thiomannosazidopyranosides    N3 OBn O

O BnO

MeOOC N3 BnO O O BnO

O

O

N3

2-Azido-3-O-benzyl-4-O-(methyl 2-azido-3-O-benzyl-2-deoxy-4-O-(4-azido-2,3-di-O-benzyl-4-deoxy-α-Dfucopyranosyl)-α/β-D-mannopyranosyluronate)-2-deoxy-β-D-1,6-anhydroglucose (24): A solution of 38 mg donor 22 (0.050 mmol) and 21 mg acceptor 23 (0.075 mmol, 1.5 equiv.) in 2 mL DCM was stirred over activated MS3Å for 30min before 17 mg N-iodosuccinimide (0.075 mmol, 1.5 equiv.) was added. The mixture was cooled to -40°C and 2 µL TMSOTf (cat.) was added. Stirring was continued and the reaction mixture was allowed to warm to rT. When TLC-analysis indicated complete reaction, 0.2 mL triethylamine was added. The reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded 32 mg of the title compound 24 (0.032 mmol, 65%, α/β=1/2) as a colorless oil. TLC: 30% EtOAc/PE; α-anomer: 1H NMR (400 MHz, CDCl3) δ = 1.20 (d, 1H, J = 6.4 Hz, H-6’’), 3.23 (s, 1H, H-2), 3.51 (s, 3H, CH3 COOMe), 3.72 (s, 1H, H-3), 3.74 – 3.82 (m, 4H, H-6, H-4’, H-5’ and H-2’’), 3.90 – 3.92 (m, 2H, H-4, H-2’), 3.91 (t, 1H, J = 3.2 Hz, H-3’’), 4.00 (dd, 1H, J = 10.0 Hz, J = 3.6 Hz, H-3’), 4.13 (d, 1H, J = 7.2 Hz, H-6), 4.19 (t, 1H, J = 4.0 Hz, H-4’’), 4.50 (d, 1H, J = 12.0 Hz, CHHPh), 4.56 (d, 1H, J = 12.0 Hz, CHHPh), 4.63 (d, 1H, J = 2.4 Hz, H-5’’), 4.66 (d, 1H, J = 12.0 Hz, CHHPh), 4.74 (d, 1H, J = 12.0 Hz, CHHPh), 4.76 (d, 1H, J = 11.6 Hz, CHHPh), 4.80 (d, 1H, J = 11.6 Hz, CHHPh), 4.81 (bs, 1H, H-1’), 4.81 – 4.83 (m, 4H, 2xCH2 Bn), 4.93 (d, 1H, J = 5.6 Hz, H-5), 5.32 (d, 1H, J = 7.6 Hz, H-1’’), 5.51 (s, 1H, H-1), 7.22 – 7.41 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 17.3 (C-6’’), 52.7 (CH3 COOMe), 59.5 (C-2 or C-2’’), 60.0 (C-2 or C-2’’), 64.5 (C-5’ or C-4’), 65.4 (C-6), 65.6 (C-4’ or C5’), 72.6 (CH2 Bn), 72.9 (CH2 Bn), 73.2 (CH2 Bn), 73.9 (CH2 Bn), 74.1 (C-5’’), 74.9 (C-4’’), 75.5, 75.9 (C-3’’, C-4 or C-2’), 76.3 (C-5), 77.5 (C-3’), 77.7 (C-3), 77.8 (C-2’ or C-4), 97.8 (C-1’’), 99.8 (C-1’), 100.6 (C-1), 129.0 – 130.5 (CH Arom), 137.0 (Cq Bn), 137.2 (Cq Bn), 137.9 (Cq Bn), 138.3 (Cq Bn), 169.3 (C=O COOMe); 13

C-GATED (100 MHz, CDCl3): 97.8 (JC1’’,H1’’ = 166 Hz, C-1’’), 99.8 (JC1’,H1’ = 167 Hz, C-1’), 100.6 (JC1,H1 = 168 Hz, C-1); β-anomer: [α]D22: +2° (c = 0.3, CHCl3); IR (neat, cm-1): 609, 1041, 1091, 1222, 1710, 2098; 1H NMR (400 MHz, CDCl3) δ = 1.19 (d, 3H, J = 6.4 Hz, H-6’’), 3.17 (s, 1H, H-2), 3.67 (dd, 1H, J = 8.7 Hz, J = 3.5 Hz, H-3’), 3.73 (s, 3H, CH3 COOMe), 3.74 – 3.77 (m, 2H, H-4’’, H-5’’), 3.78 – 3.81 (m, 2H, H-3, H-6), 3.82 (d, 1H, J = 3.8 Hz, H-2’’), 3.89 (d, 1H, J = 9.3 Hz, H-5’), 3.93 (d, 1H, J = 3.8 Hz, H-3’’), 3.96 (s, 1H, H-4), 3.98 (d, 1H, J = 3.5 Hz, H-2’), 4.15 (d, 1H, J = 7.3 Hz, H-6), 4.32 (t, 1H, J = 9.0 Hz, H-4’), 4.56 – 4.61 (m, 5H, 2xCHHPh, 3x CHHPh), 4.63 (d, 1H, J = 2.5 Hz, H-5), 4.68 (d, 1H, J = 11.8 Hz, CHHPh), 4.72 (d, 1H, J = 11.9 Hz, CHHPh), 4.79 (d, 1H, J = 11.9 Hz, CHHPh), 4.81 (s, 1H, H-1’), 5.35 (d, 1H, J = 3.8 Hz, H-1’’), 5.51 (s, 1H, H-1), 7.25 – 7.36 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 17.2 (C-6’’), 52.7 (CH3 COOMe), 58.9 (C-2), 60.6 (C-2’), 64.7 (C-4’’ or C-5’’), 64.8 (C-6), 65.2 (C-5’’ or C-4’’), 71.5 (C-5), 72.0 (CH2 Bn), 72.5 (C3’’), 72.6 (CH2 Bn), 72.8 (CH2 Bn), 73.1 (C-4’), 73.4 (CH2 Bn), 75.5 (C-2’’), 75.9 (C-5’), 77.6 (C-4), 77.8 (C-3), 79.7 (C-3’), 96.1 (C-1’), 98.2 (C-1’’), 100.9 (C-1), 127.4 – 128.5 (CH Arom), 137.2 (Cq Bn), 137.9 (Cq Bn), 138.1 (Cq Bn) 167.8 (C=O COOMe); 13C-GATED (100 MHz, CDCl3): 96.1 (JC1’,H1’ = 160 Hz, C-1’), 98.2 (JC1’’,H1’’ = 172 Hz, C-1’’), 100.9 (JC1,H1 = 175 Hz, C-1); HRMS: [M+Na]+ calcd for C47H51O12N9Na 956.35494, found 956.36039.

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Chapter 5   

References and Notes 1

Original publication: Van den Bos, L.J.; Duivenvoorden, B.A.; De Koning, M.C.; Filippov, D.V.; Overkleeft, H.S.; Van der Marel, G.A. Eur. J. Org. Chem. 2007, 116–124.

2 3

Lindberg, B. Adv. Carbohydr. Chem. Biochem. 1990, 48, 279–318. (a) Ohno, N.; Yadomae, T.; Miyazaki, T. Carbohydr. Res. 1980, 80, 297–304 (b) Jennings, H.J.; Rosell, K.G.; Carlo, D.J. Can. J. Chem. 1980, 58, 1069–1074 (c) Lugowski, C.; Romanowska, E.; Kenne, L.; Lindberg, B. Carbohydr. Res. 1983, 118, 173–181 (d) Katzenellenbogen, E.; Jennings, H.J. Carbohydr. Res. 1983, 124, 235–245 (e) Beynon, L.M.; Richards, J.C.; Perry, M.B.; Kniskern, P.J. Can. J. Chem. 1992, 70, 218–232 (f) Osa, Y.; Kaji, E.; Takahashi, K.; Hirooka, M.; Zen, S.; Lichtenthaler, F.W. Chem. Lett. 1993, 22, 1567–1570 (g) Likhosherstov, L.M.; Senchenkova, S.N.; Knirel, Y.A.; Shashkov, A.S.; Shibaev, V.N.; Stepnaya, O.A.; Kulaev, I.S. FEBS Letters 1995, 368, 113–116 (h) Osman, S.F.; Fett, W.F.; Irwin, P.; Cescutti, P.; Brouillette, J.N.; O’Connor, J.V. Carbohydr. Res. 1997, 300, 323–327 (i) Rick, P.D.; Hubbard, G.L.; Kitaoka, M.; Nagaki, H.; Kinoshita, T.; Dowd, S.; Simplaceanu, V.; Ho, C. Glycobiology 1998, 8,

4 5 6 7

557–567. (a) Reeves,, R.E. J. Am. Chem. Soc. 1950, 72, 1499–1506 (b) Litjens, R.E.J.N. Thesis Leiden University, 2005. Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348 and references cited therein. (a) Lichtenthaler, F.; Schneider-Adams, T.; Immel, S. J. Org. Chem. 1994, 59, 6735–6738 (b) Crich, D.; Dai, Z. Tetrahedron 1999, 55, 1569–1580. (a) Sato, K.I.; Yoshimoto, A. Chem. Lett. 1995, 24, 39–40 (b) Nilsson, M.; Norberg, T. J. Chem. Soc., Perk. Trans. 1 1998, 1699–1704 (c) Bousquet, E.; Khitri, E.; Lay, L.; Nicotra, F.; Panza, L.; Russo, G. Carbohydr. Res. 1998, 311, 171–181.

8 9

Paulsen, H.; Lockhoff, O. Chem. Ber. 1981, 114, 3102–3114. (a) Barresi, F.; Hindsgaul, O. Can. J. Chem. 1994, 72, 1447–1465 (b) Barresi, F.; Hindsgaul, O. Synlett 1992, 759–761 (c) Barresi, F.; Hindsgaul, O. J. Am. Chem. Soc. 1991, 113, 9376–9377. 10 (a) Crich, D.; Sun, S. J. Org. Chem. 1996, 61, 4506–4507 (b) Crich, D.; Sun, S.; Brunckova, J. J. Org. Chem. 1996, 61, 605–615. 11 Crich, D.; Sun, S. J. Am. Chem. Soc. 1998, 120, 435–436. 12 (a) Crich, D.; Sun, S. J. Org. Chem. 1997, 62, 1198–1199 (b) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020 (c) Crich, D.; Li, W.; Li, H. J. Am. Chem. Soc. 2004, 126, 15081–15086. 13 (a) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997, 119, 11217–11223 (b) Crich, D. J. Carbohydr. Chem. 2002, 21, 667–690 (c) Crich, D.; Chandrasekera, N.S. Angew. Chem. Int. Ed. 2004, 43, 5386–5389. 14 It was already suggested by Dean et al. that torsional effects in general are quite minor as compared to the electronic effects of the different hydroxyl groups: Dean, K.E.S.; Kirby, A.J.; Komarov, I.V. J. Chem. Soc., Perkin Trans. 2 2002, 337–341. 15 Jensen, H.H.; Nordstrøm, L.U.; Bols, M. J. Am. Chem. Soc. 2004, 126, 9205–9213. 16 Trichloroacetimidate: (a) Schmidt, R.R.; Toepfer, A. Tetrahedron Lett. 1991, 32, 3353–3356 (b) Weingart, R.; Schmidt, R.R.; Tetrahedron Lett. 2000, 41, 8753–8758 c) Abdel-Rahman, A.A.H.; Joke, S.; El Ashry, E.S.H.; Schmidt, R.R. Angew. Chem. Int. Ed. 2002, 41, 2972–2974. HCB-donors: Kim, K.S.; Kim, J.H.; Lee, Y.J.; Lee, Y.J.; Park, J.; J. Am. Chem. Soc. 2001, 123, 8477–8481. Dehydrative protocol: Codée, J.D.C.; Hossain, L.A.; Seeberger, P.H. Org. Lett. 2005, 7, 3251–3254. Anomeric phosphates: (a) Nagai, H.; Sasaki, K.; Matsumura, S.; Toshima, K. Carbohydr. Res. 2005, 340, 337–353 (b) Tsuda, T.; Arihara, R.; Sato, S.; Koshiba, M.; Nakamura, S.; Hashimoto, S. Tetrahedron 2005, 61, 10719–10733.

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Deactivated 1‐Thiomannosazidopyranosides    17 (a) Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279 (b) Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522. 18 (a) Litjens, R.E.J.N.; Van den Bos, L.J.; Codée, J.D.C.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Eur. J. Org. Chem. 2005, 918–924 (b) Litjens, R.E.J.N.; Den Heeten, R.; Timmer, M.S.M.; Overkleeft, H.S.; Van der Marel, G.A. Chem. Eur. J. 2005, 11, 1010–1016. 19 Crich, D.; Hutton, T.K.; Banerjee, A.; Jayalath, P.; Picione, J. Tetrahedron Asymm. 2005, 16, 105–119. 20 Srivastava, V.K.; Schuerch, C. Carbohydr. Res. 1980, 79, C13–C16 (b) Srivastava, V.K.; Schuerch, C. J. Org. Chem. 1981, 46, 1121–1126 (c) Webster, K.T.; Eby, R.; Schuerch, C. Carbohydr. Res. 1983, 123, 335– 340. 21 (a) See Chapter 4 (b) Van den Bos, L.J.; Dinkelaar, J.; Overkleeft, H.S.; Van der Marel, G.A. J. Am. Chem. Soc. 2006, 128, 13066–13067. 22 Litjens, R.E.J.N.; Leeuwenburgh, M.A.; Van der Marel, G.A.; Van Boom, J.H. Tetrahedron Lett. 2001, 42, 8693–8696. 23 (a) Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168 (b) Van den Bos, L.J.; Litjens, R.E.J.N.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2005, 7, 2007–2010. 24 Xu, L.; Price, N.P.J. Carbohydr. Res. 2004, 339, 1173–1178. 25 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323 – 326. 26 The anomeric configurations were determined by measuring 13C-GATED NMR spectra: Bock, K.; Pedersen, C. J. Chem. Soc., Perk. Trans. 2 1974, 293–299. 27 (a) Crich, D.; Vinod, A.U. Org. Lett. 2003, 5, 1297–1300 (b) Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. Tetrahedron 2004, 60, 1057–1064 (c) Codée, J.D.C.; Stubba, B.; Schiattarella, M.; Overkleeft, H.S.; Van Boeckel, C.A.A.; Van Boom, J.H.; Van der Marel, G.A. J. Am. Chem. Soc. 2005, 127, 3767–3773. 28 (a) Crich, D.; Cai, W.; Dai, Z. J. Org. Chem. 2000, 65, 1291–1297 (b) Crich, D.; Yao, Q. J. Am. Chem. Soc. 2004, 126, 8232–8236. 29 The remote participation of acyl-protection in the glucose series was recently published: Ustyuzhanina, N.; 30 31 32 33

Komarova, B.; Zlotina, N.; Krylov, V.; Gerbst, A.; Tsvetkov, Y.; Nifantiev, N. Synlett 2006, 6, 921–924. See Chapter 9. This partial donor regeneration was only observed using donor 8 and acceptor 12. Veeneman, G.H.; Van Leeuwen, S.H.; Van Boom, J.H. Tetrahedron Lett. 1990, 31, 1331–1334. (a) Dell, A.; Oates, J.; Lugowski, C.; Romanowska, E.; Kenne, B.; Lindberg, B. Carbohydr. Res. 1984, 133,

95–114 (b) Färnbäck, M.; Eriksson, L.; Senchenkova, S.; Zych, K.; Knirel, Y.A.; Sidorczyk, Z.; Widmalm, G. Angew. Chem. Int. Ed. 2003, 42, 2543–2546. 34 (a) Paulsen, H,; Lorentzen, H.P Angew. Chem., Int. Ed. Engl. 1985, 24, 773–775 (b) Paulsen, H.; Lorentzen, H.P. Carbohydr. Res. 1986, 150, 63–90. 35 Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950. 36 (a) Elchert, B.; Li, J.; Wang, J.; Rai, R.; Ptak, R.; Ward, P.; Takemoto, J.Y.; Bensaci, M.; Chang, C.-W.T.; J. Org. Chem. 2004, 69, 1513–1523 (b) Li, J.; Wang, J.; Czyryca, P.G.; Chang, H.; Orsak, T.W.; Evansons, R.; Chang, C.-W.T. Org. Lett., 2004, 6, 1381–1384. 37 No decomposition of disaccharide 22 was detected so triethylphosphite was not needed. See ref 27b. 38 Dinkelaar, J.; Witte, M.D.; Van den Bos, L.J.; Overkleeft, H.S.; Van der Marel, G.A. Carbohydr. Res. 2006, 341, 1723–1729.

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Chapter 5    39 Ganguli, A.R.S.; Coward, J.K. Tetrahedron Asymm. 2005, 16, 411–424. 40 Li, J.; Wang, J.; Czyryca, P.G.; Chang, H.; Orsak, T.W.; Evanson, R.; Chang, C.-W.T.; Org. Lett. 2004, 6, 1381–1384.

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Chapter 6 │  A Novel Strategy Towards the     Synthesis of Orthogonally       Functionalized 4‐Aminoglycosides

Abstract: A tethered nucleophilic substitution strategy for the stereoselective introduction of axially oriented amino functions on various gluco- and mannopyranosides is presented. The resulting 4-amino functionalized monosaccharide building blocks have, after some manipulations, found application in the construction of highly functionalized oligosaccharides.1

Introduction The broad variety of naturally occurring carbohydrate structures originates not only from the large number of possible interglycosidic linkages, but also from the wide diversity in monosaccharide constituents. Aminosugars,2 hexapyranoses functionalized by amino groups at different positions, represent an important category of carbohydrate units found in numerous oligosaccharides and glycoconjugates. For instance, aminosugars are not only important as essential components of bacterial capsular polysaccharides,3 but also as structural elements of aminoglycoside antibiotics.4 The biological importance of natural products containing aminosugars demands the development of efficient synthesis routes to these monosaccharides. This Chapter focuses on the stereoselective introduction of a C4 amino functionality with a cis relationship to the neighboring C5 hydroxymethyl group of hexapyranosides.5

99   

99

Chapter 6   

A convenient route to the regio- and stereoselective introduction of nitrogen substituents involves the use of tethered nitrogen nucleophiles.6 For example, allylic trichloroacetimidates have been employed in the Overman rearrangement to provide the corresponding allylic trichloroacetamides.7 Alternatively, o-iodoxybenzoic acid (IBX)8 or N-iodosuccinimide (NIS)-mediated9 cyclization of allylic trichloroacetimidates gives the corresponding transdisposed iodo-oxazolines. In an extension to these methods, this Chapter describes a novel, straightforward synthesis of orthogonally protected 4-amino glycosides. Key element in this strategy is the selective introduction of a trichloroacetimidate moiety at the C6-OH of partially protected gluco- and mannopyranosides. The hydroxyl at C4 is transformed into a suitable leaving group amenable to base-induced substitution by the tethered imidate moiety to provide the corresponding oxazine. Furthermore, after hydrolysis and suitable protection, these aminoglycosides can be readily applied as building blocks in oligosaccharide synthesis.

Results and Discussion As a first example, the transformation of methyl 2,3-di-O-benzyl-α-D-glucopyranoside (1)10 into methyl 4-acetamido-6-O-acetyl-2,3-di-O-benzyl-4-deoxy-α-D-galactopyranoside (5) was investigated (Scheme 1). Treatment of 1 with trichloroacetonitrile (Cl3CCN) in the presence of a catalytic amount of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) led to the selective formation of 6-O-acetimidate 2 in a yield of 89%. Installation of the 4-O-triflate (Tf2O, pyridine) followed by base-mediated cyclization of resulting 3 gave oxazine 4 (80% yield, 2 steps), the structure of which was fully confirmed by spectroscopic analysis. Transformation of 1 via a three-step one-pot procedure afforded oxazine 4 in slightly improved yield (89% over 3 steps). Acidic hydrolysis of 4 and acetylation of the intermediate aminoalcohol gave methyl 4-acetamido-6-O-acetyl-2,3-di-O-benzyl-4-deoxy-α-D-galactopyranoside (5) in a yield of 89%.

Scheme 1. HN OH

BnO 1

O N

O a

O

HO BnO

Cl3C

CCl3

RO BnO

O

b

OMe

2: R = H 3: R = SO2CF3

OAc O

d

O BnO

BnO

OMe

AcHN

c

BnO BnO 4

OMe

BnO

OMe

5

Reagents and conditions: (a) Cl3CCN, DBU, DCM, 0°C, 89% (b) Tf2O, pyridine, DCM (c) DiPEA, DCM, 80% (over 2 steps) (d) 1) 80% AcOH in H2O, 60°C, 2) Ac2O, pyridine, 89% (over 2 steps).

Next, diversely functionalized manno- and glucopyranosides were employed in the tethered nucleophilic galactopyranoside substitution sequence (Table 1). Subjection of methyl 2,3-di100   

100

Synthesis of 4‐Aminoglycosides    11

O-benzyl-α-D-mannopyranoside (6) to the three-step one pot procedure afforded oxazine 7, albeit in a moderate yield (entry 1). Closer inspection of the sequence of reactions revealed that the first two steps, which entailed installation of the 6-O-imidate and 4-O-triflate functions, proceeded with equal efficiency as observed for the synthesis of its glucopyranose congener 3 from diol 1 (Scheme 1). Arguably, the axially oriented 2-O-benzyl group in mannopyranoside 6 may cause 1,3-diaxial strain in the transition state hampering the formation of oxazine 7. Hydrolysis and acetylation of 7 gave fully protected 4-deoxy-4aminotaloside 8. The next objective was application of the developed protocol on protected thioglycosides, representing useful building blocks in the assembly of oligosaccharides (entries 2-4).12 Gratifyingly, ethyl 2,3-O-isopropylidene-1-thio-α-D-mannopyranoside 913 was readily transformed into oxazine 10 (entry 2). Unmasking of the oxazine ring in 10 proceeded with concomitant partial hydrolysis of the isopropylidene protective group. Acetylation and ensuing purification of the crude reaction mixture led to the isolation of 4-deoxy-4aminotalosides 11 and 12 in a combined yield of 61%. An unexpected difference in efficiency in the outcome of the three-step oxazine synthesis was observed in the case of ethyl Table 1.

entry

oxazinea

substrate Cl3C

1

HO HO BnO

O N

OBn O

BnO

HO

2

HO

O

O

O

O

O SEt

N SEt

Cl3C

HO BnO

R = Bn: 21% R = Bz: 64%

SPh

OAc OR O

R = isoprop : 40% R = Ac : 21%

AcHN

AcHN

BnO

N3 19

O

BnO

61% SEt

OBz

O O

OAc

17

N

N3 18

SEt

OR 15: R = Bn 16: R = Bz

OH O

O

BnO

OR 13: R = Bn 14: R = Bz

OMe

O

OH O

RO

64%

SEt 11: R = isoprop 12: R = Ac

10

Cl3C

4

54%

yieldc

OAc OBn O 8

AcHN O

SEt

HO BnO

BnO

O N

9

3

41%

OMe

7

Cl3C

productb AcHN

OBn O

OMe

6

yieldc

SPh

74%

BnO

OAc O

SPh

94%

N3 20

a

1) Cl3CCN, DBU, DCM, 0°C, 2) Tf2O, pyridine, DCM, 0°C, 3) DiPEA. b 1) 80% AcOH in H2O, 2) Ac2O, pyridine. c isolated yields.

101   

101

Chapter 6   

thioglucosides 1314 and 1415 (entry 3). In both cases, selective installation of the primary trichloroacetimidate proceeded well. However, treatment of the intermediate 6-O-acetimidate in 13 with triflic anhydride and pyridine followed by DiPEA-mediated cyclization afforded oxazine 15 in a rather poor yield. This probably results from the high nucleophilic nature of the anomeric thio-function. Indeed, decreasing the nucleophilicity16 of the 1-thio group by replacing the C2 benzyl protective group for a benzoyl (14), led to formation of oxazine 16 in a satisfactory yield. Analogously, application of the sequence of reactions to phenyl 2-azido3-O-benzyl-2-deoxy-1-thio-α-D-glycoside (18),17 bearing an electron withdrawing azide at C2, provided the 2,4-diamino derivative 19 in a good yield (entry 4). Both oxazines 16 and 19 were readily hydrolyzed and acetylated leading to the 4-acetamidogalactosides 17 and 20 in 61% and 94% yield, respectively. Orthogonally protected oxazine 19 was selected as a model compound to be used both as donor and acceptor in ensuing glycosylation reactions. Diaminogalactosides possessing differently functionalized amino functions are frequently encountered in natural products. For instance as 2-acetamido-4-amino-2,4,6-trideoxy-galactopyranosides present in the capsular zwitterionic polysaccharides of Bacteroides fragilis.3 The group of Van Boom and Van der Marel recently demonstrated18 that partially protected thioglycosides can be effectively employed as acceptor glycosides in the dehydrative condensation procedure developed by Gin and coworkers.19 Aglycon 21 was readily obtained by acidic hydrolysis of oxazine 19 and subsequent selective reaction of the amine with N-(benzyloxycarbonyl-oxy)succinimide (Scheme 2). Dehydrative condensation was accomplished by activating 2,3,4,6-tetra-Obenzyl-α/β-D-galactopyranose (23)20 under the influence of diphenylsulfonium bistriflate, followed by addition of acceptor 21 to afford the α-linked thiodisaccharide 24 in 65% yield. This excellent anomeric selectivity is surprising as reactive donors in combination with primary alcohol acceptors usually give anomeric mixtures.21 Next, the fully orthogonally protected thiodonor 22 was prepared by acetylation of compound 21. Activation of this disarmed16 donor 22 was accomplished by treatment with diphenylsulfoxide (Ph2SO)/triflic anhydride (Tf2O),22 which was shown to be a more potent thiophilic promoter system than the analogous benzenesulfinyl piperidine (BSP)/Tf2O23 combination recently developed by the Crich-laboratory. Addition of methyl glucoside 25,24 to the activated donor, smoothly afforded disaccharide 26 as an anomeric mixture (α/β = 1/1) in excellent yield.25

Conclusion This Chapter describes a new, straightforward synthesis procedure for the construction of 4amino-4-deoxyglycosides, and their application as donor and acceptor in condensation reactions towards disaccharides. It was found that nucleophilic inversion of C4 in the glucoseries proceeded considerably more efficiently than inversion at C4 in the manno-series. Most 102   

102

Synthesis of 4‐Aminoglycosides   

likely, 1,3-diaxial interaction hampers the incoming nucleophile leading to a reduced yield. Retention of configuration was not observed in the presented cases. Furthermore, diaminogalactoside 22 showed promise as a valuable building block in the Ph2SO/Tf2O-mediated construction of larger oligosaccharide structures.

Scheme 2. 19 OH CbzHN

OAc

BzO OMe 25

O

BnO

26

N3 O BzO BzO

O

d

BnO

a

O

BzO BzO

OBn O

BnO CbzHN BnO

BnO 23

OR O

SPh

N3

BzO OMe b

21: R = H 22: R = Ac

OH

BnO

OBn

SPh

O

BnO

c

BnO 24

O

N3

O

CbzHN

OBn

Reagents and conditions: (a) 1) 80% AcOH in H2O, 60°C, 2) CbzOSu, pyridine, 95% (over 2 steps) (b) Ac2O, pyridine, quant. (c) 23, Ph2SO, Tf2O, TTBP, DCM, -60°C then 21, 65% (d) 22, Ph2SO, Tf2O, DCM, -60°C then 25, 95% (α/β = 1/1).

Experimental Section General procedure 1H and 13C NMR spectra were recorded on a Jeol JNM-FX-200 (200/50.1 MHz) and a Bruker AV-400 (400/100 MHz) spectrometer. Chemical shifts are given in ppm (δ) relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and Q-Star Applied Biosystems QTOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide and TTBP were removed by co-evaporation with toluene. TTBP was synthesized as described by Crich and coworkers.26 Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled immediately prior to use. Solvents used for flash chromatography and TLC were of technical grade and distilled before use. Flash chromatography was performed on Baker silica gel (0.063 – 0.200 mm). TLC-analysis was conducted on DC-fertigfolien (Schleicher & Schuell, F1500, LS254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulphuric acid followed by charring at ~150°C. Free amine functions were detected by spraying with a ninhydrine-solution in EtOH followed by charring at ~150°C. Oxazine 4: A solution of methyl 2,3-di-O-benzyl-α-D-glucopyranoside (1) (0.5 g; 1 mmol) in 5 mL DCM was cooled to 0°C under an Ar-atmosphere. Subsequently, 0.1 mL trichloroacetonitrile (1.05 mmol, 1.05 equiv.) and 3 µL 1,8-diazabicyclo[5.4.0]undec-7-ene

Cl3C O N O BnO

(0.01 mmol, 0.01 equiv.) were added to the solution. According to TLC-analysis, all starting material was consumed after 5min. Subsequent treatment of the reaction mixture with 0.4 mL pyridine (5 mmol, 5 equiv.) and 0.24 mL trifluoromethanesulfonic anhydride (1.5 mmol, 1.5 equiv.) afforded the triflated product in 5min at 0°C. Base-induced cyclization was accomplished by addition of 1.0 mL diisopropylethylamine (5 mmol, 5 equiv.) and stirring was allowed for 3½h. The reaction mixture was purified BnO

OMe

103   

103

Chapter 6    by flash chromatography (20% EtOAc/PE). Removal of the eluent gave 0.40 g of the title compound 4 (0.80 mmol, 80%) as a yellow oil. TLC: 35% EtOAc in PE; [α]D22: +63° (c = 1, CHCl3); IR (cm-1): 1001, 1028, 1043, 1089, 1195, 1220, 1352, 1454, 1496, 1510, 1679, 1718; 1H NMR (400 MHz, CDCl3): δ = 3.37 (s, 3H, CH3 OMe), 3.62 (dd, 1H, J = 3.5 Hz, J = 10.1 Hz, H-2), 3.94 (m, 1H, H-4), 4.04 (bd, 1H, J = 2.0 Hz, H-5), 4.13 (dd, 1H, J = 10.1 Hz, J = 4.4 Hz, H-3), 4.29 (d, 1H, J = 11.8 Hz, H-6), 4.42 (dd, 1H, J = 11.8, J = 1.3 Hz, H-6), 4.60 (d, 1H, J = 3.5 Hz, H-1), 4.65 (d, 1H, J = 11.8 Hz, CHHPh); 4.77 – 4.88 (m, 3H, CHHPh, CH2 Bn), 7.25 – 7.47 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 54.1 (C-4), 55.7 (Me), 60.4 (C-5), 69.2 (C-6), 71.8 (CH2 Bn), 73.8 (CH2 Bn), 75.2 (C-3), 75.6 (C-2), 99.5 (C-1), 127.4 – 128.3 (CH Arom), 138.3 (Cq Bn), 138.9 (Cq Bn), 153.0 (C=NH); ESI-MS: 500.0 [M+H]+. Methyl 4-acetamido-6-O-acetyl-2,3-di-O-benzyl-4-deoxy-α-D-galactopyranose (5): A solution of 0.44 g oxazine 4 (0.89 mmol) in 5 mL 80% AcOH/H2O was stirred for 30min at ambient BnO OMe temperature. After removal of the solvent under reduced pressure, the residue was coevaporated several times with toluene. The crude product was subsequently dissolved in 4 mL pyridine and AcHN

OAc O

BnO

treated with 1 mL Ac2O. Stirring was continued for 8h before the solvent was removed in vacuo. After coevaporation with toluene the crude mixture was purified by flash chromatography (30% EtOAc/PE). Removal of the eluent gave 0.49 g of the title compound 5 (0.79 mmol, 89%) as a yellow oil. TLC: 35% EtOAc/PE; [α]D22: +2° (c = 1, CHCl3); IR: 1028, 1099, 1155, 1195, 1232, 1369, 1651, 1739; 1H NMR (400 MHz, CDCl3): δ = 2.04 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 3.38 (s, 3H, CH3 OMe), 3.45 – 3.49 (dd, 1H, J = 4.0 Hz, J = 10.1 Hz, H2), 3.96 – 4.00 (dd, 1H, J = 10.7 Hz, J = 4.7 Hz, H-3), 4.08 – 4.15 (m, 3H, H-5, H-6, H-6), 4.52 (d, 1H, J = 11.1 Hz, CHHPh), 4.64 (d, 1H, J = 4.0 Hz, H-1), 4.65 (d, 1H, J = 12.2 Hz, CHHPh), 4.77 (t, 1H, J = 4.4 Hz, H-4), 4.78 (d, 1H, J = 11.1 Hz, CHHPh), 4.86 (d, 1H, J = 12.2 Hz, CHHPh), 5.60 (d, 1H, J = 10.1 Hz, NH), 7.26 – 7.39 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 20.8 (CH3 Ac), 23.5 (CH3 Ac), 47.6 (C-4), 55.4 (CH3 OMe), 63.2 (C-6), 66.7 (C-5), 71.7 (CH2 Bn), 73.6 (CH2 Bn), 75.3 (C-2), 76.0 (C-3), 98.6 (C-1), 127.7 – 128.4 (CH Arom), 137.9 (Cq Bn), 138.2 (Cq Bn), 170.5 (C=O); HRMS: [M+H]+ calcd for C15H34NO7 458.2101, found 458.2097. HN

CCl3

Methyl 2,3-di-O-benzyl-6-O-trichloroacetimidate-α-D-glucopyranoside (2): To a stirred

solution of 1.79 g compound 1 (5 mmol) in 25 mL DCM at 0°C was added 0.5 mL trichloroacetonitrile (5 mmol, 1 equiv.) and 5 µL 1,8-diazabicyclo[5.4.0]undec-7-ene (0.05 mmol, 0.05 equiv.). Stirring was allowed for 45min after which the reaction mixture was BnO OMe concentrated (waterbath ~30°C). Flash chromatography (20% EtOAc/PE) and removal of the eluent gave 2.23 g. of the title compound 2 (4.31 mmol, 89%) as a colorless oil. TLC: 50% EtOAc/PE. 1H NMR O

O

HO BnO

(400 MHz, CDCl3): δ = 3.39 (s, 3H, CH3 OMe), 3.46 (dd, 1H, J = 9.0 Hz, H-4), 3.50 (dd, 1H, J = 9.6 Hz, J = 3.6 Hz, H-2), 3.82 (t, 1H, J = 9.2 Hz, H-3), 3.87 (m, 1H, J = 10.1 Hz, J = 5.0 Hz, H-5), 4.48 (d, 1H, J = 11.8 Hz, H6), 4.60 (d, 1H, J = 3.4 Hz, H-1), 4.61 (dd, 1H, J = 11.9 Hz, J = 5.0 Hz, H-6), 4.64 (d, 1H, J = 11.9 Hz, CHHPh), 4.76 (d, 2H, J = 11.6 Hz, 2xCHHPh), 4.97 (d, 1H, J = 11.3 Hz, CHHPh), 7.29 – 7.37 (m, 10H, H Arom), 8.23 (bs, 1H, NH); 13C NMR (100 MHz, CDCl3): δ = 55.1 (CH3 OMe), 68.5 (C-6), 69.6 (C-5), 70.0 (C-4), 73.2 (CH2 Bn), 75.6 (CH2 Bn), 79.6 (C-2), 81.0 (C-3), 98.1 (C-1), 127.8 – 128.5 (CH Arom), 137.9 (Cq Bn), 138.6 (Cq Bn); ESI: 518.1 [M+H]+, 540.0 [M+Na]+. Cl3C

BnO

Oxazine 7: Oxazine 7 was obtained from compound 6 via the same procedure as described

O N

OBn O OMe

104   

104

for the conversion of compound 1 into 4. The reaction mixture was purified by flash chromatography (15% EtOAc/PE). Removal of the eluent gave 75 mg of the title compound 7 (0.15 mmol, 41%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ = 3.38 (s, 3H, CH3

Synthesis of 4‐Aminoglycosides    OMe), 3.66 (t, 1H, J = 2.6 Hz, H-2), 4.01 (m, 2H, J = 2.6 Hz, J = 4.7 Hz, H-4, H-5), 4.06 (dd, 1H, J = 4.6 Hz, J = 2.6 Hz, H-3), 4.28 (d, 1H, J = 11.2 Hz, H-6), 4.54 (dd, 1H, J = 11.5 Hz, J = 2.6 Hz, H-6), 4.66 (d, 2H, J = 12.3 Hz, 2xCHHPh), 4.78 (d, 1H, J = 12.0 Hz, CHHPh), 4.85 (d, 1H, J = 2.7 Hz, H-1), 4.86 (d, 1H, J = 12.5 Hz, CHHPh), 7.31 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 52.4 (C-4), 55.4 (CH3 OMe), 61.4 (C-5), 68.7 (C-6), 70.9 (CH2 Bn), 72.7 (CH2 Bn), 74.1 (C-2, C-3), 101.0 (C-1), 127.6 – 128.4 (CH Arom), 138.3 (Cq Bn), 138.9 (Cq Bn), 151.8 (C=N); ESI-MS: 500.0 [M+H]+, 522.1 [M+Na]+. Methyl 4-acetamido-6-O-acetyl-2,3-di-O-benzyl-4-deoxy-α-D-talopyranoside (8): Compound 8 was prepared from oxazine 7 via the same procedure as described for the conversion of

OAc OBn O

AcHN BnO

compound 4 into 5. The reaction mixture was purified by flash chromatography (20% EtOAc/PE). Removal of the eluent gave 43 mg of the title compound 8 (0.096 mmol, 64%) as a yellow oil. TLC: 40% EtOAc/PE; [α]D22: +36° (c = 0.66, CHCl3); IR (cm-1): 1041, 1114, 1230, 1369, 1454, 1515, 1670, 1739; 1H NMR (400 MHz, CDCl3): δ = 1.81 (s, 3H, NHAc), 2.06 (s, 3H, CH3 Ac), 3.34 (s, 3H, CH3 OMe), 3.76 (m, 1H, J = 1.6 Hz, H-2), 3.81 (dd, 1H, J = 4.2 Hz, J = 3.1 Hz, H-3), 3.97 (m, 1H, H-5), 4.12 (dd, OMe

1H, J = 8.0 Hz, J = 3.6 Hz, H-6), 4.24 (dd, 1H, J = 11.7 Hz, J = 4.2 Hz, H-6), 4.43 (d, 1H, J = 11.6 Hz, CHHPh), 4.66 (m, 1H, H-4), 4.68 (d, 2H, J = 11.2 Hz, 2xCHHPh), 4.76 (d, 1H, J = 11.0 Hz, CHHPh), 4.81 (d, 1H, J = 1.2 Hz, H-1), 6.95 (d, 1H, J = 9.3 Hz, NH), 7.30 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 20.8 (CH3 Ac), 23.2 (CH3 Ac), 46.5 (C-4), 54.8 (CH3 OMe), 64.0 (C-6), 68.2 (C-5), 69.9 (CH2 Bn), 72.1 (C-3), 74.1 (CH2 Bn), 76.2 (C-2), 99.9 (C-1), 127.5 – 128.4 (CH Arom), 137.6 (Cq Bn), 137.8 (Cq Bn), 170.7 (C=O); ; HRMS: [M+H]+ calcd for C15H34NO7 458.2101, found 458.2174. Cl3C

Oxazine 10: Oxazine 10 was obtained from compound 9 via the same procedure as described for the conversion of compound 1 into 4. The reaction mixture was purified by flash

O N

O

O

chromatography (10% EtOAc/PE). Removal of the eluent gave 216 mg of the title compound 10 (0.55 mmol, 54%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ = 1.23 (t, 3H, J = 7.4 Hz, CH3 SEt), 1.27 (s, 3H, CH3 isoprop), 1.47 (s, 3H, CH3 isoprop), 2.62 (m, 2H, CH2 SEt), 4.19 (d, 1H, J = 7.2 Hz, H-2), 4.23 (m, 1H, H-4), 4.27 (s, 1H, H-5), 4.28 (m, 1H, H-6), 4.35 (m, 1H, H-6), 4.67 (dd, 1H, J = 7.2 Hz, J = 3.7 Hz, H-3), 5.07 (s, 1H, H-1); 13C NMR (100 MHz, CDCl3): δ = 14.7 (CH3 SEt), 24.9 (CH3 isoprop), 25.4 O

SEt

(CH2 SEt), 26.1 (CH3 isoprop), 49.5 (C-4), 61.1 (C-5), 67.2 (C-6), 72.0 (C-2), 76.0 (C-3), 81.4 (C-1), 110.4 (Cq isoprop), 154.1 (C=N); ESI-MS: 390 [M+H]+, 412 [M+Na]+.

AcHN O

Ethyl 4-acetamido-6-O-acetyl-4-deoxy-2,3-O-isopropylidene-1-thio-α-D-talopyranoside (11): Compound 11 was prepared from oxazine 10 via the same procedure as described for the

OAc O O

conversion of compound 4 into 5. The reaction mixture was purified by column chromatography (EtOAc). Removal of the eluent gave 77 mg of the title compound 11 (0.22 mmol, 40%) as a yellow oil, together with 50 mg ethyl 4-acetamido-2,3,6-tri-O-acetyl-4-deoxy-1-thio-α-D-talopyranoside 12 (0.13 mmol, 21%). TLC: 40% EtOAc/PE; [α]D22: +142° (c = 1); IR (cm-1): 1002, 1037, 1083, 1211, 1235, 1373, 1519, 1666, 1739; 1H NMR (400 MHz, CDCl3): δ = 1.32 (t, 3H, J = 7.4 Hz, CH3 SEt), 1.34 (s, 3H, CH3 isoprop), SEt

1.50 (s, 3H, CH3 isoprop), 2.04 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 2.57 (m, 1H, CHH SEt), 2.74 (m, 1H, CHH SEt), 4.05 (dd, 1H, J = 5.9 Hz, J = 1.0 Hz, H-2), 4.10 (dd, 1H, J = 11.8 Hz, J = 4.1 Hz, H-6), 4.23 (d, 1H, J = 11.8 Hz, H-6), 4.34 (t, 1H, J = 5.9 Hz, H-3), 4.38 (m, 1H, H-5), 4.47 (m, 1H, H-4), 5.52 (s, 1H, H-1), 5.86 (d, 1H, J = 9.9 Hz, NH); 13C NMR (100 MHz, CDCl3): δ = 15.1 (CH3 SEt), 21.6 (CH3 Ac), 24.3 (CH3 Ac), 24.8 (CH2 SEt), 26.2 (CH3 isoprop), 27.1 (CH3 isoprop), 46.5 (C-4), 64.1 (C-6), 67.5 (C-5), 72.2 (C-3), 74.6 (C-2), 80.1 (C-1), 110.0 (Cq isoprop), 170.5 (C=O), 171.3 (C=O); HRMS: [M+H]+ calcd for C15H27NO6S 348.1403, found 348.1434. Ethyl 4-acetamido-2,3,6-tri-O-acetyl-4-deoxy-1-thio-α-D-tallopyranoside 12: TLC: 40%

105   

105

Chapter 6    EtOAc/PE. 1H NMR (400 MHz, CDCl3): δ = 1.30 (t, 3H, J = 7.4 Hz, CH3 SEt), 2.00 (s, 3H, CH3 Ac), 2.04 (s, 3H, CH3 Ac), 2.07 (s, 3H, CH3 Ac), 2.20 (s, 3H, CH3 Ac), 2.66 (m, 2H, CH2 SEt), 4.12 (dd, 1H, J = 11.8 Hz, J = 4.6 Hz, H-6), 4.22 (dd, 1H, J = 11.7 Hz, J = 7.7 Hz, H-6), 4.59 (dd, 1H, J = 10.3 Hz, J = 3.6 Hz, H-4), 4.65, (m, 1H, H-5), 5.17 (t, 1H, J = 3.6 Hz, H-3), 5.21 (m, 1H, J = 3.5 Hz, H-2), 5.31 (s, 1H, H-1), 6.19 (d, 1H, J = 10.1 Hz, NH), 13C NMR (100 MHz, CDCl3): δ = 14.5 (CH3 SEt), 20.5 (CH3 Ac), 20.6 (CH3 Ac), 21.0 (CH3 Ac), 23.2 (CH3 Ac), 24.9 (CH2 SEt), 46.9 (C-4), 62.8 (C-6), 65.7 (C-2), 68.2 (C-5), 70.6 (C-3), 82.5 (C-1), 168.7 (C=O), 169.3 (C=O), 169.9 (C=O), 170.3 (C=O); ESI-MS: 414 [M+Na]+. Oxazine 15: Oxazine 15 was obtained from compound 13 via the same procedure as

Cl3C

O

described for the conversion of compound 1 into 4. The reaction mixture was purified by flash chromatography (20% EtOAc/PE). Removal of the eluent gave 63 mg of the title BnO SEt OBn compound 15 (0.12 mmol, 21%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ = 1.23 (t, 3H, J = 7.6 Hz, CH3 SEt), 2.60 – 2.70 (m, 2H, J = 7.5 Hz, CH2 SEt), 3.55 (t, 1H, J = 9.2 Hz, H-2), 3.75 (dd, 1H, J = 8.9 Hz, J = 4.3 Hz, H-3), 3.83 (bs, 1H, H-5), 3.93 (bs, 1H, H-4), 4.32 (d, 1H, J = 11.7 Hz, H-6), 4.39 (d, 1H, N

O

J = 9.4 Hz, H-1), 4.55 (d, 1H, J = 10.2 Hz, H-6), 4.78 – 4.89 (m, 4H, 2xCH2 Bn), 7.25 – 7.44 (m, 10H, H Arom); C NMR (100 MHz, CDCl3): δ = 15.0 (CH3 SEt), 22.9 (CH2 SEt), 53.3 (C-4), 67.4 (C-5), 67.4 (C-6), 69.4 (CH2 Bn), 71.6 (CH2 Bn), 76.6 (C-2), 80.3 (C-3), 83.9 (C-1), 127.6 – 133.0 (CH Arom), 138.1 (Cq Bn), 138.2 (Cq Bn), 154.4 (C=N); ESI-MS: 532.1 [M+H]+, 554.1 [M+Na]+.

13

Cl3C

O N O

BnO

SEt

OBz

Oxazine 16: Oxazine 16 was obtained from compound 14 via the same procedure as described for the conversion of compound 1 into 4. The reaction mixture was purified by flash chromatography (20% EtOAc/PE). Removal of the eluent gave 63 mg of the title compound 16 (0.12 mmol, 64%) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ = 1.19 (t,

3H, J = 7.5 Hz, CH3 SEt), 2.67 – 2.75 (m, 2H, J = 7.5 Hz, CH2 SEt), 3.90 – 3.93 (dd, 1H, J = 9.4 Hz, J = 4.2 Hz, H-3), 3.96 (d, 1H, J = 2.1 Hz, H-5), 4.06 (bs, 1H, H-4), 4.37 (d, 1H, J = 11.7 Hz, H-6), 4.52 (d, 1H, J1,2 = 9.5 Hz, H-1), 4.62 (dd, 1H, J = 11.7 Hz, J = 1.6 Hz, H-6), 4.70 – 4.80 (dd, 2H, J = 12.8 Hz, CH2Ph), 5.40 (t, 1H, J = 9.5 Hz, J = 9.4 Hz, H-3), 7.18 – 8.03 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 14.7 (CH3 SEt), 22.1 (CH2 SEt), 53.1 (C-4), 67.8 (C-5), 68.7 (C-2), 69.3 (C-6), 70.9 (CH2 Bn), 77.3 (C-3), 82.5 (C-1), 127.6 – 133.0 (CH Arom), 129.9 (Cq Bz), 137.7 (Cq Bn), 165.0 (C=N); ESI: 544.1 [M+H]+. AcHN BnO

OAc O OBz

SEt

Ethyl 4-acetamido-6-O-acetyl-2-O-benzoyl-3-O-benzyl-4-deoxy-1-thio-β-D-galactopyranoside (17): Compound 17 was prepared from oxazine 16 via the same procedure as described for the conversion of compound 4 into 5. The reaction mixture was purified by flash

chromatography (45% EtOAc/PE). Removal of the eluent gave 33 mg of the title compound 17 (0.066 mmol, 61%) as a yellow oil. TLC: 35% EtOAc/PE; [α]D22: +14° (c = 0.42, CHCl3); IR (cm-1): 1242, 1369, 1454, 1550, 1647, 1720; 1H NMR (400 MHz, CDCl3): δ = 1.24 (t, 3H, J = 7.4 Hz, CH3 SEt), 2.07 (s, 3H, CH3 Ac), 2.08 (s, 3H, CH3 Ac), 2.65 – 2.71 (m, 2H, J = 7.4 Hz, CH2 SEt), 3.74 (dd, 1H, J = 9.7 Hz, J = 4.6 Hz, H-3), 3.86 (dd, 1H, J = 6.3 Hz, J = 1.2 Hz, H-5), 4.21 (d, 2H, J = 6.3 Hz, H-6), 4.46 (d, 1H, J = 12.6 Hz, CHHPh), 4.56 (d, 1H, J = 10.1 Hz, H-1), 4.70 (d, 1H, J = 12.6 Hz, CHHPh), 4.89 – 4.92 (m, 1H, H-4), 5.22 (t, 1H., J = 9.9 Hz, H-2), 5.96 (d, 1H, J = 10.1 Hz, NH), 7.09 – 8.01 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 14.9 (CH3 SEt), 20.8 (CH3 Ac), 23.3 (CH3 Ac), 25.2 (CH2 SEt), 46.6 (C-4), 63.0 (C-6), 69.9 (C-2), 70.4 (CH2 Bn), 76.0 (C-5), 76.6 (C-3), 84.8 (C-1), 127.6 – 133.2 (CH Arom), 129.5 (Cq Bz), 137.2 (Cq Bn), 170.4 (C=O), 170.7 (C=O); HRMS: [M+H]+ calcd for C26H33NO7S 502.1821, found 502.1828.

106   

106

Synthesis of 4‐Aminoglycosides    Cl3C

O N O

BnO

SPh

N3

Oxazine 19: Oxazine 19 was obtained from compound 18 via the same procedure as described for the conversion of compound 1 into 4. The reaction mixture was purified by flash chromatography (30% EtOAc/PE). Removal of the eluent gave 4.16 g of the title compound 19 (8.2 mmol, 74%) as a yellow oil. TLC: 40% EtOAc/PE; [α]D22: +46° (c = 1,

CHCl3); IR (cm-1): 1172, 1226, 1678, 2110; 1H NMR (400 MHz, CDCl3): δ = 3.37 (t, 1H, J = 9.8 Hz, H-2), 3.62 (dd, 1H, J = 9.6 Hz, J = 4.4 Hz, H-3), 3.83 (dd, 1H, J = 3.7 Hz, J = 1.5 Hz, H-5), 3.88 (d, 1H, J = 4.1 Hz, H-4), 4.35 (m, 2H, J = 10.0 Hz, H-1, H-6), 4.62 (dd, 1H, J = 12.16 Hz, H-6), 4.72 (d, 1H, J = 12.1 Hz, CHHPh), 4.80 (d, 1H, J = 12.1 Hz, CHHPh), 7.26 – 7.55 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 51.8 (C-4), 60.0 (C-5), 68.0 (C-2), 69.4 (C-6), 71.2 (CH2 Bn), 79.1 (C-3), 85.4 (C-1), 127.9 – 134.2 (CH Arom), 129.5 (Cq SPh), 137.4 (Cq Bn), 153.7 (C=N); ; ESI-MS: 513.4 [M+H]+, 535.0 [M+Na]+. AcHN BnO

OAc O

SPh

N3

Phenyl 4-acetamido-6-O-acetyl-2-azido-3-O-benzyl-2,4-dideoxy-1-thio-β-D-galactopyranoside (20): Compound 20 was prepared from oxazine 19 via the same procedure as described for the conversion of compound 4 into 5. The reaction mixture was purified by flash chromatography

(45% EtOAc/PE). Removal of the eluent gave 1.06 g of the title compound 20 (2.27 mmol, 94%) as a yellow oil. TLC: 40% EtOAc/PE; [α]D22: -22° (c = 0.24, CHCl3); IR (cm-1): 1037, 1103, 1230, 1365, 1438, 1535, 1654, 1739, 2110; 1H NMR (400 MHz, CDCl3): δ = 1.96 (s, 3H, CH3 Ac), 2.06 (s, 3H, CH3 Ac), 3.27 (1H, t, J = 10.0 Hz, H-2), 3.53 (dd, 1H, J = 9.7 Hz, J = 4.2 Hz, H-3), 3.74 (dd, 1H, J = 7.0 Hz, J = 5.0 Hz, H-5), 4.09 – 4.23 (m, 2H, H-6), 4.38 (d, 1H, J = 10.3 Hz, H-1), 4.50 (d, 1H, J = 10.8 Hz, CHHPh), 4.75 (d, 1H, J = 6.9 Hz, H-4), 4.78 (d, 1H, J = 10.8 Hz, CHHPh), 5.43 (d, 1H, J = 10.1 Hz, NH), 7.30 – 7.59 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 20.7 (CH3 Ac), 23.3 (CH3 Ac), 45.7 (C-4), 61.2 (C-2), 62.8 (C-6), 71.4 (CH2 Bn), 75.7 (C-5), 79.4 (C-3), 86.1 (C-1), 128.1 – 133.8 (CH Arom), 130.9 (Cq SPh), 136.6 (Cq Bn), 170.3 (C=O); HRMS: [M+H]+ calcd for C23H28N4O5S 471.1624, found 471.1745. Phenyl 4-(N-benzyloxycarbonyl)-amino-2-azido-3-O-benzyl-2,4-dideoxy-1-thio-β-D-galactopyranoside (21): Oxazine 19 (0.81 g, 1.58 mmol) was stirred in 5 mL 80% AcOH in H2O at N3 ambient temperature. After 15min, the clear solution was diluted with toluene, concentrated and co-evaporated several times with toluene. The crude oil was dissolved in 5 mL DCM followed by addition

CbzHN BnO

OH

O

SPh

of 0.26 mL NEt3 (2.0 mmol, 1.25 equiv.) and 0.4 g CbzOSu (1.6 mmol, 1.01 equiv.). The reaction mixture was stirred for 8h at room temperature before EtOAc was added. The mixture was washed with 50 mL 1M HCl. The water-layer was extracted twice with 50 mL EtOAc. The combined organic layers were dried (MgSO4), filtered en concentrated in vacuo. Flash chromatography (50% EtOAc/PE) and removal of the eluent afforded 844 mg of the title compound 21 (1.50 mmol, 95%) as a colorless oil. TLC: 50 % EtOAc/PE; [α]D22: -6° (c = 1, CHCl3); IR (cm-1): 1056, 1215, 1257, 1512, 1705, 2110; 1H NMR (200 MHz, CDCl3): δ = 3.01 (bs, 1H, OH), 3.24 (t, 1H, J = 9.9 Hz, H-2), 3.57 (m, 3H, H-3, H-6), 3.72 (m, 1H, H-5), 4.36 (m, 2H, J = 10.2 Hz, H-1, H-4), 4.50 (d, 1H J = 10.9 Hz, CHHPh), 4.68 (d, 1H, J = 11.3 Hz, CHHPh), 4.83 (d, 1H, J = 7.8 Hz, NH), 5.12 (s, 2H, CH2 Cbz), 13C NMR (50.4 MHz, CDCl3): δ = 47.5 (C-4), 60.5 (C-6), 61.1 (C-2), 67.0 (CH2 Cbz), 70.8 (CH2 Bn), 77.9 (C-5), 78.8 (C-3), 86.1 (C-1), 127.6 – 132.6 (CH Arom), 131.3 (Cq SPh), 135.8 (Cq Cbz), 136.6 (Cq Bn), 157.3 (C=O); HRMS: [M+H]+ calcd for C27H30N4O5S 521.1780, found 521.1829. CbzHN BnO

OAc O

SPh

Phenyl 6-O-acetyl-4-(N-benzyloxycarbonyl)-amino-2-azido-3-O-benzyl-2,4-dideoxy-1thio-β-D-galactopyranoside (22): Compound 21 (593 mg, 1.14 mmol) was dissolved in 5

N3 mL pyridine followed by the addition of 2.5 mL Ac2O. The reaction mixture was stirred for 2h at room temperature. The mixture was concentrated and co-evaporated several times with toluene. After flash chromatography (30% EtOAc/PE) and removal of the eluent, 606 mg of the title compound 22 (1.14 mmol,

107   

107

Chapter 6    quant.) was obtained as a colorless oil. TLC: 50 % EtOAc/PE; [α]D22: -13° (c = 1, CHCl3); IR (cm-1): 1037, 1103, 1226, 1365, 1454, 1510, 1716, 2110; 1H NMR (200 MHz, CDCl3): δ = 2.04 (s, 3H, CH3 Ac), 3.27 (t, 1H, J = 10.2 Hz, J = 9.7 Hz, H-2), 3.51 (dd, 1H, J = 9.9 Hz, J = 4.3 Hz, H-3), 3.71 (t, 1H, J = 5.8 Hz, H-5), 4.20 (m, 2H, J = 5.1 Hz, H-6), 4.34 (d, 1H, J = 10.2 Hz, CHHPh), 4.42 (m, 1H, H-4), 4.52 (d, 1H, J = 11.0 Hz, CHHPh), 4.83 (d, 2H, J = 10.6 Hz, H-1, NH), 5.08 (s, 2H, CH2 Cbz), 7.25 – 7.54 (m, 10H, H Arom); 13C NMR (50.4 MHz, CDCl3): δ = 20.7 (Ac), 48.0 (C-4), 61.2 (C-2), 62.9 (C-6), 67.2 (CH2 Cbz), 71.4 (CH2 Bn), 75.6 (C-5), 79.6 (C3), 86.3 (C-1), 127.9 – 133.4 (CH Arom), 131.1 (Cq SPh), 136.0 (Cq Cbz), 136.7 (Cq Bn), 156.2 (C=O Cbz), 170.5 (C=O Ac); ESI-MS: 563.3 [M+H]+, 585.0 [M+Na]+. BnO BnO

OBn

SPh

O BnO

O

N3

O

CbzHN

OBn

Phenyl 4-(N-benzyloxycarbonyl)-amino-2-azido-3-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-2,4-dideoxy-1-thio-β-D-galactopyranoside (24): A solution of 54 mg donor 23 (0.1 mmol), 40 mg Ph2SO (0.2 mmol, 2 equiv.) and 74 mg tri-tertbutylpyrimidine (0.3 mmol, 3 equiv.) in 4 mL DCM was stirred over 100 mg activated MS4Å for 30min The mixture was cooled to -78°C and 26 µL triflic acid anhydride

(0.15 mmol, 1.5 equiv.) was added. The mixture was allowed to warm to -40°C in 1h followed by addition of 52 mg acceptor 21 (0.1 mmol, 1 equiv.) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to 0°C. Subsequently, 0.16 mL NEt3 (2 mmol, 20 equiv.) was added. Flash chromatography (20% EtOAc/PE) and removal of the eluent afforded 66 mg of the title compound 24 (65 µmol, 65%) as a yellow oil. TLC: 50% EtOAc/PE; [α]D22: +13° (c = 1, CHCl3); IR (cm-1): 1030, 1096, 1215, 1454, 1496, 1716, 2110; 1H NMR (400 MHz, CDCl3): δ = 3.18 (t, 1H, J = 10.0 Hz, H-2’), 3.45 (m, 2H, J = 6.7 Hz, J = 9.7 Hz, H-3, H-6), 3.53 (dd, 1H, J = 9.3 Hz, J = 6.4 Hz, H-6), 3.59 (q, 1H, J = 5.9 Hz, H-6’), 3.75 (m, 2H, H-5’, H-6’), 3.83 (dd, 1H, J = 10.0 Hz, J = 2.7 Hz, H-3), 3.89 (s, 1H, H-4), 3.95 (t, 1H, J = 6.3 Hz, H-5), 4.02 (dd, 1H, J = 10.1 Hz, J = 3.6 Hz, H-2), 4.32 (d, 1H, J = 10.0 Hz, H-1’), 4.38 (s, 1H, H-4’), 4.40 (m 2H, CH2 Bn), 4.56 (d, 1H, J = 11.4 Hz, CHHPh), 4.69 (t, 2H, J = 13.0 Hz, CH2 Bn), 4.80 (m, 3H, CHHPh), CH2 Bn), 4.81 (d, 1H, J = 3.2 Hz, H-1’), 4.92 (d, 1H, J = 11.4 Hz, CHHPh), 5.06 (dd, 2H, J = 12.2 Hz, CH2 Cbz), 7.20 – 7.37 (m, 35H, H Arom); 13C NMR (100 MHz, CDCl3): δ = 48.5 (C-4’), 61.2 (C-3’), 67.0 (CH2 Cbz), 67.6 (C-6’), 69.1 (C-6), 69.4 (C-5’), 71.3 (CH2 Bn), 73.1 (CH2 Bn), 73.4 (CH2 Bn), 73.5 (CH2 Bn), 74.7 (CH2 Bn), 74.9 (C-4), 76.5 (C-2), 76.7 (C-5), 78.9 (C-3), 79.8 (C-2’), 86.2 (C-1’), 98.2 (C-1), 124.7 – 133.2 (CH Arom), 131.5 (Cq SPh), 136.1 – 138.7 (Cq Bn), 156.3 (C=O); HRMS: [M+H]+ calcd for C61H64N4O10S 1043.4259, found 1043, 4280. CbzHN BnO

OAc O N3 O BzO BzO

O

Methyl 2,3,4-tri-O-benzoyl-6-O-(6-O-acetyl-4-(N-benzyloxycarbonyl)-amino-2 azido-3-O-benzyl-2,4-dideoxy-α/β-D-galactopyranosyl)-α-D-glucopyranoside (26): A solution of 53 mg donor 22 (0.1 mmol) and 25 mg Ph2SO (0.12 mmol, 1.2

BzO OMe

equiv.) in 3 mL DCM was stirred over 100 mg activated MS4Å for 30min The mixture was cooled to -78°C before 22 µL triflic acid anhydride (0.13 mmol, 1.3 equiv.) was added. The mixture was stirred for 5min followed by addition of 75 mg acceptor 25 (0.1 mmol, 1 equiv.) in 1 mL DCM. The mixture was allowed to warm to 0°C. Subsequently, 0.16 mL NEt3 (2 mmol, 20 equiv.) was added. Flash chromatography (30% EtOAc/PE) and removal of the eluent afforded 92 mg of the title compound 26 (95 µmol, 95%) as a colorless oil. TLC: 50% EtOAc/PE; IR (cm-1): 1026, 1091, 1245, 1450, 1519, 1724, 2110; 1H NMR (400 MHz, CDCl3): δ = 4.27 (d, J = 8.0 Hz, H-1’ β-anomer), 4.90 (d, J = 3.4 Hz, H-1’ α-anomer), 4.27 (d, J = 3.5 Hz, H-1); 13C NMR (100,8 MHz, CDCl3): δ = 96.8 (C-1), 97.9 (C-1’ α-anomer), 102.5 (C-1’ β-anomer); HRMS: [M+H]+ calcd for C51H52N4O15 959.3345, found 959.3394.

108   

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Synthesis of 4‐Aminoglycosides   

References and Notes 1

Original publication: Van den Bos, L.J.; Codée, J.D.C.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel,

3

G.A. Org. Biomol. Chem. 2003, 1, 4160–4165. (a) Banoub, J.; Boullanger, P.; Lafont, D. Chem. Rev. 1992, 92, 1167–1195 (b) Bernfield, M.; Gotte, M.; Park, O.; Reizes, O.; Fitsgerald, M.L.; Lincecum, J.; Zako, M. Annu. Rev. Biochem. 1999, 68, 729–777 (c) Dwek, R.A. Chem. Rev. 1996, 96, 683–720. (a) Wang, Y.; Kalka-Moll, W.M.; Roehrl, M.H.; Kasper, D.L. Proc. Natl. Acad. Sci. USA 2000, 97, 13478–

4

13483 (b) Choi, Y.-H.; Roehrl, M.H.; Kasper, D.L.; Wang, J.Y. Biochemistry 2002, 41, 15144–15151 (c) Kalka-Moll, W.M.; Tzianabos, A.O.; Bryant, P.W.; Niemeyer, N.; Ploegh, H.L.; Kasper, D.L. J. Immunol. 2002, 169, 6149–6153. For a recent review on aminoglycoside antibiotics see: (a) Kotra, L.P.; Mobashery, S. Curr. Org. Chem.

2

5

6

2001, 5, 193–205 (b) Michael, K.; Tor, Y. Chem. Eur. J. 1998, 4, 2091–2098 (c) Sears, P.; Wong, C.-H. Angew. Chem. Int. Ed. 1999, 38, 2300–2324. (a) Smid, P.; Jörning, W.P.A; Van Duuren, A.M.G.; Boons, G.-J.; Van der Marel, G.A.; Van Boom, J.H. J. Carbohydr. Chem. 1992, 11, 849–865 (b) Hermans, J.P.G.; Elie, C.J.J.; Van der Marel, G.A.; Van Boom, J.H. J. Carbohydr. Chem. 1987, 6, 451–462. For a comprehensive review: Knapp, S. Chem. Soc. Rev. 1999, 28, 61–72; See for related studies employing inversion on carbamates as the key step: (a) Knapp, S.; Kukkola, P.J.; Sharma, S.; Dhar T.G.M.; Naughton, A.B.J. J. Org. Chem. 1990, 55, 5700–5710 (b) Semmelhack, M.F.; Jiang, Y.; Ho, D. Org. Lett. 2001, 3, 2403–2406; Churchill, D.G.; Rojas, C.M. Tetrahedron Lett. 2002, 43, 7225–7228 (c) Bates, R.W.; Sa-Ei, K. Org. Lett. 2002, 4, 4225–4227.

7

(a) Cardillo, G.; Orena, M.; Sandri, S.; Tomasini, C. J. Org. Chem. 1984, 49, 3951–3953 (b) Overman, L.E. J. Am. Chem. Soc. 1978, 98, 2901–2910. 8 Nicolaou, K.C.; Baran, P.S.; Zhong, Y.-L.; Vega, J.A. Angew. Chem. Int. Ed. 2000, 39, 2525–2529. 9 Sammes, P. G.; Thetford, D. J. Chem. Soc., Perk. Trans. 1 1988, 111–123. 10 Bernotas, R.C. Tetrahedron Lett. 1990, 31, 469–472. 11 Winnik, F.M.; Brisson, J.-R.; Carver, J.P.; Kripinsky, J.J. Carbohydr. Res. 1982, 103, 15–28. 12 Garegg, P.J. Adv. Carbohydr. Chem. Biochem. 1997, 52, 179–205. 13 Zegelaar-Jaarsveld, K.; Duynstee, H.I.; Van der Marel, G.A.; Van Boom, J.H. Tetrahedron 1996, 52, 3575– 3592. 14 Nakagawa, T.; Ueno, K.; Kashiwa, M.; Watanabe, J. Tetrahedron Lett. 1994, 35, 1921–1924. 15 Zegelaar-Jaarsveld, K.; Smits, S.A.W.; Van der Marel, G.A.; Van Boom, J.H. Bioorg. Med. Chem. 1996, 4, 1819–1832. 16 Based on the “armed-disarmed” principle first introduced by Fraser-Reid: Mootoo, D.R.; Konradsson, P.; Udodong, U.; Fraser-Reid, B. J. Am. Chem. Soc. 1988, 110, 5583–5584. 17 Compound 18 was prepared by de-acetalization of phenyl 2-azido-3-O-benzyl-4,6-O-benzylidene-4-deoxy1-thio-α-D-glucoside. Martin-Lomas, M.; Flores-Mosquera, M.; Chiara, J.L. Eur. J. Org. Chem. 2000, 8, 1547–1562. 18 Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950. 19 (a) Garcia, B.A.; Poole, J.L.; Gin, D.Y. J. Am. Chem. Soc. 1997, 119, 7597–7598 (b) Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279. 20 Marco-Contelles, J.; Gallego, P.; Rodriguez-Fernandez, M.; Khiar, N.; Destabel, C. J. Org. Chem. 1997, 62, 7397–7412.

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Chapter 6    21 Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155–224. 22 Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522. 23 Crich, D.; and Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. 24 Byramova, N.E.; Ovchinnikov, M.V.; Backinowsky, L.V.; Kochetkov, N.K. Carbohydr. Res. 1983, 124, C8–C11. 25 Addition of the base tri-tert-butylpyrimidine (TTBP), as prescribed in the literature, gave rise to the formation of unexpected N-sulfated byproducts. See ref 22. 26 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326.

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Chapter 7│ Synthesis of a Protected       Trisaccharide Repeating Unit of the    Zwitterionic Polysaccharide Sp1

Abstract: This Chapter describes a convergent route of synthesis for the protected repeating unit trisaccharide structure 2 of the zwitterionic polysaccharide Sp1, using 1-hydroxyl and 1-thio functionalized donor and acceptor building blocks. Furthermore, a new synthesis approach for the demanding 2,4-diamino fucose residue 4 is presented.

Introduction Capsular polysaccharides (CPs) are major components of the bacterial cell wall and represent the virulence factors of many pathogens.1 Most of these CPs are neutral or negatively charged and are, in general, considered as T-cell independent antigens.2 Interestingly, the groups of Kasper and Tzianabos reported that CPs with a zwitterionic charge motif, show CD4+ T cell responses.3 One example is the polysaccharide found on the cell wall of the Streptococcus pneumoniae species, which plays a key role in the development of intraabdominal sepsis.4 The repeating unit of this zwitterionic CP, called Sp1, has one positive and two negatively charged groups per repeating unit (1, Figure 1).5 Structurefunction studies revealed that the dual charge motif on the carbohydrate core is at the basis of this unusual immunological activity.6,7 In this Chapter the chemical synthesis of the protected repeating unit structure 2 of the zwitterionic polysaccharide Sp1 is described using 1-hydroxyl8 and 1-thioglycoside9 donors in

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combination with the diphenylsulfoxide (Ph2SO)/triflic anhydride (Tf2O) activator system (Figure 1). The target trisaccharide structure 2 comprises two galacturonic acid moieties (3 and 5)10 and the uncommon, orthogonally functionalized 2,4-diamino fucose residue 4. A new route of synthesis for residue 4 was examined using the tethered nucleophilic inversion approach described in Chapter 6.11 Lönn and Lönngren previously reported on the synthesis of the non-reducing end [D-galacturonic acid-α-(1→3)-2-acetamido-4-amino-1-methyl-α-Dfucosamine] dimer.12

Figure 1. O HO O

COO

BnO

Cl O NH3

HO

HO

O

O

AcHN HO

BnO O

O 3

O

O

COOMe O

BnO OBn

HO 1

2

1st glycosylation NHTCA

O

BnO

O N3

COO

SPh

O

COOMe O NHTCA

O OBn

HO N3 4

OMe

OTBS 2nd glycosylation HO

COOMe O

BnO

OMe

OBn 5

Results and Discussion The galacturonic acid building blocks 3 and 5 were obtained starting from 4,6-diol 7 and 3,6-diol 9 (Scheme 1). Diol 7 was prepared from thcompound 6 using literature procedures.13 Diol 9 was efficiently synthesized from the known galactoside 814 in a three-step/one-pot procedure. Regioselective silylation15 of both C3 and C6 using TBDMSCl and imidazole in dichloromethane was followed by benzylation of the residual C2 and C4 hydroxyl groups. TLC-analysis of the reaction mixture only showed minor side-product formation indicating negligible silyl migration.16 Acidic removal of the silyl protection afforded the desired building block 9 in a yield of 53% over the 3 steps. In the next step, diols 7 and 9 were chemo- and regioselectively oxidized using the TEMPO/BAIB reagent combination to afford galacturonic acid 1010 and lactone 3.17 Treatment of the crude acid 10 with freshly prepared diazomethane afforded terminal acceptor species 5 in a yield of 57% over the 2 steps. The formation of lactone derivative 3 can be rationalized by initial formation of lactol derivative 11 followed by further oxidation towards lactone 3. Several procedures to 2,4-diamino fucose derivatives, in which both amino groups are protected in the same manner, have been published.18,19 Methods dealing with the synthesis of orthogonally protected 2,4-diamino fucose residues commence with regioselective deoxygenation at C6 of 2-acetamido20 or 2-phthalimido21 functionalized D-glucosamine and

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112

Zwitterionic Polysaccharide Sp1    Scheme 1. HO HO BnO

R4O

COOR O

d OMe

O

R3O

from 7

10: R=H 5: R=Me

a b

R1

O SPh

O

c

SPh

O

c

O

BnO

from 9

OR2

OBn d

OR5

O

BnO OBn

6: R1=β-OMe, R2=R3=R4=R5=H 7: R1=β-OMe, R2=R3=Bn, R4=R5=H 8: R1=β-SPh, R2=R3=R4=R5=H 9: R1=β-SPh, R2=R4=Bn, R3=R5=H

OBn

11

3

Reagents and conditions: (a) see ref. 13 (b) 1) TBDMSCl, Imidazole, DMF, 2) BnBr, NaH, DMF, 0°C, 3) TsOH, MeOH, reflux, 53% (over 3 steps) (c) TEMPO, BAIB, DCM, H2O, 75% (for 3) (d) 1) TEMPO, BAIB, DCM/H2O (2/1), 2) CH2N2, DMF, 57% (over 2 steps).

ensuing nucleophilic displacement of the equatorially oriented OH4 by an azide nucleophile. The presence of a C2 participating group in these derivatives prevents their use as α-selective donor glycosides and therefore a different approach to suitably protected derivative 4 was devised starting from 2-azido glucose 1322 (Scheme 2).23 The first goal to be addressed is the regioselective de-oxygenation of the C6 position of compound 13, which was easily obtained from glucosamine hydrochloride 12,22 in the presence of the sensitive azide functionality. So, regioselective tosylation of compound 13 afforded compound 14 in a yield of 80% along with minor formation of 3,6-di-O-tosylated by-product. Displacement of the C6 tosyl functionality by reaction with NaI in refluxing butanone (Finkelstein conditions) led to the isolation of diol 15 in excellent yield. Sodium borohydride reduction of the intermediate C6 iodo-function proceeded selectively and gave quinovosamine derivate 16 in a yield of 76%, with almost no products arising from azide reduction. Attempts to directly reduce the C6 tosyl function in compound 14 using NaBH4 resulted in prolonged reaction times and decreased reaction efficiencies compared to iodinated compound 15. In the next step, the regioselective introduction of the axially oriented C4 amino function was addressed by means of a one-pot tethered nucleophilic inversion approach presented in Chapter 6.11 Treatment of compound 16 with an equimolar amount of trichloroacetonitrile (Cl3CCN) and a catalytic amount of DBU resulted in the predominant formation of the 3-O-trichloroacetimidate intermediate. After triflation of OH4, excess DiPEA was added to afford the labile oxazoline derivative 17. Mild acid treatment of the crude reaction mixture selectively cleaved the oxazoline functionality in Scheme 2. OH

a

O

HO HO Cl

H3 N 12

R HO HO

OH b c d

e

O OTBS N3 13: R = OH 14: R = OTos 15: R = I 16: R = H

TCAHN

N

Cl3C

f

O O

OTBS

O HO

OTBS N3

N3 17

4

Reagents and conditions: (a) see ref. 22 (b) TsCl, pyridine, 80% (c) NaI, butanone, reflux, 96% (d) NaBH4, DMSO, 76% (e) Cl3CCN, DBU, DCM, 0°C then Tf2O, pyridine then DiPEA (f) Amberlite IR120 H+, MeOH, 38% (over 4 steps).

113   

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compound 17 yielded the 4-N-trichloroacetamide target compound 4. Two-dimensional 1H NMR spectroscopy nicely confirmed the inverted chirality at the C4 position. The assembly of the target trisaccharide 22 started at the non-reducing end using lactone building block 3 as donor (Scheme 3). Pre-activation of lactone 3 using in situ generated diphenylsulfonium bistriflate and subsequent treatment with partially protected acceptor 4 yielded α-coupled disaccharide 18 in 74% yield.24 Liberation of the anomeric hydroxyl group was accomplished by treatment of disaccharide 18 with NEt3·3HF in THF giving hemiacetal donor 19. Disarmed disaccharide 19 was then condensed with uronate acceptor 5 under influence of the Ph2SO/Tf2O activation system to give trisaccharide 20, fully αstereoselective. For purification reasons, trisaccharide 20 was processed further by acidmediated opening of the lactone bridge giving trisaccharide 2 in a yield of 58% over the last 2 steps. Scheme 3. O O

O SPh

O

3

O

a

O

BnO

TCAHN O O

BnO

b

c

O N3

OBn

TCAHN O

BnO

O O

O

N3 OBn

OR

OBn 18: R = β-OTBS 19: R = α/β-OH

20

d O

COOMe O

BnO

2

OMe

OBn

Reagents and conditions: (a) 3, Ph2SO, Tf2O, TTBP, DCM, -60°C then 4, 74% (b) NEt3·3HF, THF, 99% (c) 19, Ph2SO, Tf2O, DCM, -40°C then 5 (d) cat. H2SO4, MeOH, 58% (over 2 steps).

With the trisaccharide 2 in hand, a global deprotection strategy was investigated as outlined in Figure 2. The most challenging goal in this respect is maintaining orthogonality between both amine functionalities at the middle fucose residue. The first attempt involves initial cleavage of all base labile groups (methyl esters and trichloroacetamide), followed by reprotection of the liberated C4’ amine with a tert-butyloxycarbonyl (Boc) group. It was found that base treatment proceeded well using KOH in aqueous THF. However, ensuing reprotection of the C4’ amine group went troublesome and only a minor amount of Bocprotected amine was detected, most probably due to steric hindrance. In the next attempt, the C2’ azide functionality was first converted into an acetamide using neat thiolacetic acid, Figure 2.

1

114   

114

H2, Pd/C then Ac2O then H+

incomplete Boc-protection

1) KOH, THF/H2O 2) Boc2O, NaHCO3, H2O/dioxane

2

1) AcSH (neat) 2) KOH, THF/H 2O

formation of smaller fragments

H2, Pd/C

1

Zwitterionic Polysaccharide Sp1    25

followed by basic hydrolysis of both methyl ester and trichloroacetamide groups. Unfortunately, base treatment resulted predominantly in smaller fragments with minor methyl ester cleavage. These smaller fragments most probably originate from C4-C5 (β-)elimination at the galacturonic acid residues.26

Conclusion This Chapter describes the synthesis of the protected trisaccharide repeating unit of the zwitterionic polysaccharide Sp1 using 1-thio and 1-hydroxyl glycosides in combination with the Ph2SO/Tf2O activator system. A new route of synthesis towards the demanding, orthogonally protected 2,4-diamino fucose residue is presented. Furthermore, 1thiogalacturonic acid ester and its corresponding lactone are efficiently employed in the synthesis. It became clear that the combination of galacto-configured uronic acid derivatives and sterically encumbered N-trichloroacetamides should be circumvented in future research to minimize unwanted side-reactions during deprotection. Changing the 2,4-diamino fucose to the terminal position and/or replacing the N-trichloroacetyl protection for a Nbenzyloxycarbonyl group will probably prevent these problems. On the other hand, the application of lactone functionalized donor glycosides reduces the number of synthetic steps at the monosaccharide and oligosaccharide level. Cleavage of the lactone bridge in trisaccharide 20 forms a rapid entry into larger oligosaccharide constructs enabling future structural and biological studies.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Brüker DMX-400 and a Brüker AV-400 (400/100 MHz), Brüker AV500 (500/125 MHz) and a Brüker DMX-600 (600/150 MHz) spectrometer. Chemical shifts (δ) are given in ppm relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and Q-Star Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide and TTBP were removed by co-evaporation with toluene. TTBP was synthesized as described by Crich et al.27 Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography were of pro analysi quality. Flash chromatography was performed on Fluka silica gel 60 (0.04 – 0.063 mm). TLC-analysis was conducted on DC-alufolien (Merck, Kieselgel60, F254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in H2O

115   

115

Chapter 7    followed by charring at ~150°C. All reactions were performed under an inert atmosphere of Argon unless stated otherwise.

BnO

Phenyl 2,4-di-O-benzyl-1-thio-β-D-galactopyranoside (9): To a cooled (0°C) solution of 2.72 g

OH

compound 814 (10.0 mmol) in 50 mL DMF was added 3.24 g TBSCl (21.5 mmol, 2.15 equiv.) and 3.18 g imidazole (21.5 mmol, 2.15 equiv.). The mixture was allowed to warm to room OBn temperature. After stirring for 8h the reaction was quenched with MeOH, taken up in EtOAc and washed with H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude product was filtered through a plug of silica gel using (10% EtOAc/PE) as the eluent. After evaporation of the O

HO

SPh

solvent, the crude product was dissolved in 50 mL DMF and cooled to 0°C before 3 mL BnBr (25 mmol, 2.5 equiv.) and 1 g NaH (60% in min. oil, 25 mmol, 2.5 equiv.) were added. The mixture was allowed to stir for 12 h before the reaction was quenched by addition of MeOH, taken up in Et2O and washed with H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The oily substance was suspended in 50 mL MeOH and a catalytic amount of TsOH was added. The mixture was refluxed until TLC indicated complete conversion. After addition of 5 mL NEt3 the reaction mixture was concentrated under reduced pressure. Flash chromatography afforded 2.39 g of the title compound 9 (5.26 mmol, 53%) as a colorless oil. TLC: 50% EtOAc/PE; [α]D22: +1° (c = 1.4, CHCl3); IR (neat, cm-1): 694, 640, 732, 871, 1018, 1049, 1311; 1H NMR (500 MHz, CDCl3) δ = 1.86 (bs, 1H, OH), 2.33 (bs, 1H, OH), 3.49 (t, 1H, J = 6.5 Hz, J = 5.5 Hz, H-5), 3.56 – 3.58 (m, 1H, H-6), 3.71 (bs, 1H, H-3), 3.74 (t, 1H, J = 9.5 Hz, H-2), 3.79 (s, 1H, H-4), 3.85 (dd, 1H, J = 11.5 Hz, J = 7.0, H-6), 4.61 (d, 1H, J = 9.5 Hz, H-1), 4.62 (d, 1H, J = 12.0 Hz, CHHPh), 4.64 (d, 1H, J = 11.0 Hz, CHHPh), 4.77 (d, 1H, J = 12.0 Hz, CHHPh), 4.92 (d, 1H, J = 11.0 Hz, CHHPh), 7.27 – 7.37 (m, 15H, H Arom); 13C NMR (125 MHz, CDCl3) δ = 62.2 (C-6), 74.8 (CH2 Bn), 75.3 (CH2 Bn), 75.7 (C-4), 75.9 (C-3), 78.2 (C-2), 79.0 (C-5), 98.3 (C-1), 127.2 – 128.5 (CH Arom), 133.8 (Cq SPh), 137.9 (Cq Bn), 138.1 (Cq Bn); HRMS: [M+Na]+ calcd for C26H28O5SNa 475.15497, found 475.15567. O SPh

O BnO

O

Phenyl 2,4-di-O-benzyl-1-thio-β-D-galactopyranosidurono-6,3-lactone (3): To a vigorously stirred solution of 1.19 g compound 9 (2.63 mmol) in 10 mL DCM and 3 mL H2O was added 82 mg TEMPO (0.5 mmol, 0.2 equiv.) and 2.12 g BAIB (6.58 mmol, 2.5 equiv.). Stirring was

allowed until TLC indicated complete conversion of the starting material to a higher running OBn spot (~15min). The reaction mixture was quenched by the addition of 50 mL Na2S2O3 solution (10% in H2O). The mixture was then extracted twice with EtOAc (100 mL) and the combined organic phase was dried (MgSO4), filtered and concentrated. Flash column chromatography using EtOAc/petroleum ether afforded 883 mg of the title compound 3 (1.97 mmol, 75%) as a colorless oil. TLC: 20% EtOAc/PE; [α]D22: -95° (c = 0.5, CHCl3); IR (neat, cm-1): 979, 1026, 1060, 1097, 1151, 1365, 1454, 1799, 2869, 3030; 1H NMR (400 MHz, CDCl3) δ = 4.02 (s, 1H, H-4), 4.23 (d, 1H, J = 4.8 Hz, H-2), 4.36 (s, 1H, H-5), 4.48 (d, 1H, J = 11.8 Hz, CHHPh), 4.51 (s, 2H, CH2 Bn), 4.56 (d, 1H, J = 11.8 Hz, CHHPh), 4.79 (d, 1H, J = 4.4 Hz, H-3), 5.40 (s, 1H, H1), 7.14 – 7.43 (m, 15H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 70.6 (C-4), 71.1 (CH2 Bn), 72.7 (CH2 Bn), 75.7 (C-5), 78.4 (C-2), 78.6 (C-3), 85.6 (C-1), 127.6 – 132.4 (CH Arom), 133.5 (Cq SPh), 136.4 (Cq Bn), 136.5 (Cq Bn), 172.5 (C=O); HRMS: [M+NH4]+ calcd for C26H28O5SN 466.16882, found 466.16946. HO BnO

COOMe O

OMe

Methyl (methyl 2,3-di-O-benzyl-β-D-galactopyranoside) uronate (5): Compound 5 was obtained from known methyl 2,3-di-O-benzyl galactopyranoside 713 following a two-step

BnO procedure. First, acid 10 was obtained in the same way as described for the conversion of diol 9 into lactone 3. This intermediate free acid 10 was directly processed further by treatment with freshly prepared diazomethane28 [caution: CH2N2 is reported to be explosive] affording 877 mg of the title compound 5

116   

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Zwitterionic Polysaccharide Sp1    -1

mmol, 57% over 2 steps) as white, amorphous crystals. TLC: 25% EtOAc/PE; IR (neat, cm ): 694, 732, 894, 1041, 1080, 1265, 1357, 1442, 1720, 2947; 1H NMR (400 MHz, CDCl3) δ = 2.55 (bs, 1H, OH-4), 3.56 (dd, 1H, J = 9.2 Hz, J = 3.2 Hz, H-3), 3.60 (s, 3H, CH3 OMe), 3.67 (dd, 1H, J = 9.2 Hz, J = 7.6 Hz, H-2), 3.83 (s, 3H, CH3 COOMe), 4.06 (d, 1H, J = 1.2 Hz, H-5), 4.28 (d, 1H, J = 7.6 Hz, H-1), 4.32 (dd, 1H, J = 3.2 Hz, J = 1.2 Hz, H4), 4.69 (d, 1H, J = 12.0 Hz, CHHPh), 4.71 (d, 1H, J = 11.2 Hz, CHHPh), 4.76 (d, 1H, J = 12.0 Hz, CHHPh), 4.88 (d, 1H, J = 11.2 Hz, CHHPh), 7.26 – 8.01 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 52.6 (CH3 COOMe), 57.2 (CH3 OMe), 67.9 (C-4), 72.5 (CH2 Bn), 73.6 (C-5), 75.1 (CH2 Bn), 78.2 (C-2), 79.7 (C-3), 104.5 (C-1), 127.6 – 128.5 (CH Arom), 137.5 (Cq Bn), 138.4 (Cq Bn), 168.4 (C=O); HRMS: [M+Na]+ calcd for C22H26O7Na 425.15545, found 425.15707. Tert-butyldimethylsilyl 2-azido-2-deoxy-6-O-tosyl-β-D-glucopyranoside (14): A solution of 3.8 g of compound 13 (13.1 mmol) in 65 mL anhydrous pyridine was cooled to 0˚C N3 followed by the addition of 3.0 g tosylchloride (16 mmol, 1.2 equiv). The reaction mixture was stirred for 5h under Ar-atmosphere at room temperature. The reaction was quenched by addition of ice-cold OTos O

HO HO

OTBS

MeOH. The mixture was diluted with 100 mL EtOAc and washed with 50 mL aq. 1M HCl solution and 50 mL sat. aq. NaHCO3 solution. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 4.7 g of the title compound 14 (10.4 mmol, 80%) as a colorless oil. TLC: 70% EtOAc/PE; IR (neat, cm-1): 686, 779, 833, 1072, 1180, 1249, 1369, 2106; 1H NMR (500 MHz, CDCl3) δ = 0.13 (s, 3H, CH3 TBDMS), 0.15 (s, 3H, CH3 TBDMS), 0.91 (s, 9H, tBu, TBDMS), 2.45 (s, 3H, CH3 Ts), 3.17 (bs, 2H, 2xOH), 3.19 (dd, 1H, J = 10.0 Hz, J = 7.6 Hz, H-2), 3.31 (dd, 1H, J = 10.0 Hz, J = 7.6 Hz, H-3), 3.42 – 3.50 (m, 2H, H-4 and H-5), 4.21 (dd, 1H, J = 10.8 Hz, J = 4.4 Hz, H-6), 4.27 (d, 1H, J = 10.0 Hz, H-6), 4.53 (d, 1H, J = 7.6 Hz, H-1), 7.33 (d, 2H, J = 8.0 Hz, 2H Arom), 7.79 (d, 2H, J = 8.0 Hz, 2H Arom); 13C NMR (100 MHz, CDCl3) δ = -5.41 (CH3 TBDMS), -4.47 (CH3 TBDMS), 17.8 (Cq tBu TBDMS), 21.5 (CH3 Ts), 25.4 (tBu TBDMS), 68.7 (C-6), 67.8, 69.6, 73.2, 74.2 (C-2, C-3, C-4 and C-5), 96.9 (C-1), 127.8, 129.8 (CH Arom), 132.4 (Cq Ts), 144.9 (Cq Ts); HRMS: [M+Na]+ calcd for C19H31O7N3SiNa 496.15442, found 496.15455. Tert-butyldimethylsilyl 2-azido-2,6-dideoxy-6-iodo-β-D-glucopyranoside (15): Compound

I O

14 (3.7 g, 8.9 mmol) was dissolved in 50 mL butanone before 3.0 g NaI (20 mmol, 2.2 N3 equiv.) was added. The mixture was refluxed for 5h. The reaction mixture was cooled to ambient temperature, diluted with 100 mL EtOAc and washed with 50 mL aq. 1M Na2S2O3 solution and 50 mL H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 3.7 g of the title compound 15 (8.5 mmol, 96%) as HO HO

OTBS

a yellow oil. TLC: 70% EtOAc/PE; [α]D22: +45° (c = 1.5, CHCl3); IR (neat, cm-1): 686, 779, 833, 1072, 1180, 1249, 2106; 1H NMR (400 MHz, CDCl3) δ = 0.21 (s, 3H, CH3 TBDMS), 0.23 (s, 3H, CH3 TBDMS), 0.95 (s, 9H, tBu TBDMS), 3.19 (m, 1H, H-5), 3.23 (t, 1H, J = 8.8 Hz, J = 8.0 Hz, H-2), 3.31 (dd, 1H, J = 10.8 Hz, J = 2.4 Hz, H-6), 3.34 – 3.39 (m, 2H, J = 8.8 Hz, J = 6.8 Hz, H-3 and H-4), 3.57 (dd, 1H, J = 10.8 Hz, J = 2.0 Hz, H6), 3.83 (bs, 2H, 2xOH), 4.64 (d, 1H, J = 7.2 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ = -5.2 (CH3 TBDMS), 3.9 (CH3 TBDMS), 5.0 (C-6), 17.8 (Cq tBu TBDMS), 25.6 (tBu TBDMS), 68.4 (C-2), 73.8 (C-3 or C-4), 74.1 (C-4 or C-3), 74.9 (C-5), 97.0 (C-1); HRMS: [M+NH4]+ calcd for C12H28N4O4SiI 447.09190, found 447.09271.

HO HO

O

Tert-butyldimethylsilyl 2-azido-2,6-dideoxy-β-D-glucopyranoside (16): To a stirred

solution of 3.7 g compound 15 (8.6 mmol) in 100 mL DMSO was added 1.9 g NaBH4 (50 mmol, 5.8 equiv.). The reaction mixture was stirred for 2h, quenched by MeOH and separated between EtOAc/H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced OTBS

N3

117   

117

Chapter 7    pressure. The crude oil was redissolved in 75 mL DMSO and 0.5 g NaBH4 (13.1 mmol, 1.5 equiv.) was added. Stirring was continued for 6h followed by addition of excess methanol. The mixture was diluted with 100 mL EtOAc and washed with 50 mL aq. 1M HCl solution and 50 mL sat. aq. NaHCO3 solution. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 2.0 g of the title compound 16 (6.6 mmol, 76%) as a colorless oil. TLC: 70% EtOAc/PE; [α]D22: +35° (c = 2.8 CHCl3); IR (neat, cm-1): 686, 779, 833, 1010, 1064, 1180, 1249, 2106; 1H NMR (400 MHz, CDCl3) δ = 0.14 (s, 6H, 2xCH3 TBDMS), 0.93 (s, 9H, tBu TBDMS), 1.32 (d, 1H, J = 6.1 Hz, H-6), 2.71 (bs, 2H, 2xOH), 3.22 – 3.34 (m, 4H, H-2, H-3, H-4, H-5), 4.56 (d, 1H, J = 7.0 Hz, H-1); 13C NMR (100 MHz, CDCl3) δ = -5.22 (CH3 TBDMS), -4.32 (CH3 TBDMS), 17.6 (C-6), 17.9 (Cq tBu TBDMS), 25.6 (tBu TBDMS), 69.6, 71.6, 74.3, 74.7 (C-2, C-3, C-4 and C-5), 96.9 (C-1); HRMS: [M+NH4]+ calcd for C12H29O4N4Si 321.19526, found 321.19549. TCAHN O

OTBS

HO

Tert-butyldimethylsilyl 4-trichloracetamido-2-azido-2,4,6-trideoxy-β-D-galactopyranoside (4): Diol 16 (151 mg, 0.5 mmol) and 60 µL Cl3CCN (0.6 mmol, 1.2 equiv.) were dissolved

N3 in 4 mL DCM, stirred over MS3Ǻ and cooled to -13°C. Subsequently a catalytic amount of DBU was added. The reaction mixture was allowed to stir for 1h and cooled down to -30°C. Then 0.2 mL pyridine (2.5 mmol, 5 equiv.) and 99 µL triflic anhydride (0.6 mmol, 1.2 equiv.) were added. The reaction mixture was allowed to warm to room temperature and stirred for 30min. followed by the addition of 0.8 mL

DiPEA (5 mmol, 10 equiv.). After 4h the reaction mixture was diluted with 100 mL EtOAc and successively washed with 1M HCl (50 mL) and sat. aq. NaHCO3 (50 mL). The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude dark red oil [oxazoline 17: TLC: 30% EtOAc/PE; ESI-MS: 429.0 [M+Na]+; HRMS: [M+H]+ calcd for C14H23O3N4Cl3Si 429.06778, found 429.06811.] was suspended in 2.5 mL MeOH and Amberlite IR120 H+-resin was added until pH = 5. Stirring was continued for 15min. before the reaction mixture was filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 84 mg (0.19 mmol, 38% over 4 steps) compound 4 as a yellow oil. TLC: 30% EtOAc/PE; [α]D22: -11° (c = 0.4 CHCl3); IR (neat, cm-1): 678, 779, 825, 1049, 1110, 1180, 1257, 1512, 1712, 2113; 1H NMR (400 MHz, CDCl3) δ = 0.16 (s, 6H, 2xCH3 TBDMS), 0.96 (s, 9H, tBu TBDMS), 1.24 (d, 3H, J = 6.4 Hz, C-6), 3.16 (bs, 1H, OH-3), 3.18 (dd, 1H, J = 10.6 Hz, J = 7.6 Hz, H-2), 3.72 (dd, 1H, J = 10.4 Hz, J = 4.4 Hz, H-3), 3.77 (dq, 1H, J = 7.2 Hz, J = 6.4 Hz, J = 1.2 Hz, H-5), 4.23 (ddd, 1H, J = 9.4 Hz, J = 4.4 Hz, J = 1.2 Hz, H-4), 4.53 (d, 1H, J = 8.0 Hz, H-1), 6.89 (d, 1H, J = 9.2 Hz, NH-4); 13C NMR (100 MHz, CDCl3) δ = -5.1 (CH3 TBDMS), -4.4 (CH3 TBDMS), 16.6 (C-6), 17.9 (Cq tBu TBDMS), 25.5 (tBu TBDMS), 55.0 (C-4), 66.5 (C-2), 69.3 (C-5), 71.4 (C-3), 92.4 (Cq CCl3), 97.4 (C-1), 163.5 (C=O TCA); HRMS: [M+H]+ calcd for C14H26O4N4SiCl3 447.07834, found 447.08124. O

TCAHN O

BnO

O O

O

N3 OBn

OTBS

Tert-butyldimethylsilyl 2-azido-3-O-(2,4-di-O-benzyl-α-D-galactopyranosylurono-6,3-lactone)-4-trichloracetamido 2,4,6-trideoxy-β-D-galactopyranoside (18): A solution of 314 mg lactone 3 (0.7 mmol), 202 mg diphenylsulfoxide (1.0 mmol, 1.4 equiv.) and 521 mg tri-tert-butylpyrimidine (2.1 mmol, 3

equiv.) in 10 mL DCM was stirred over activated MS3Å for 30min. The mixture was cooled to -60°C before 166 µL triflic acid anhydride (1.0 mmol, 1.4 equiv.) was added. The mixture was allowed to warm to -50°C in 15min followed by addition of 290 mg acceptor 4 (0.65 mmol, 0.9 equiv.) in 5 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to -10°C. The reaction mixture was diluted with 50 mL EtOAc and washed with water (50 mL). The aqueous phase was extracted twice with EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography (EtOAc/PE) and removal of the eluent afforded the 406 mg of the title compound 18 (0.52 mmol, 74%) as a colorless oil. TLC:

118   

118

Zwitterionic Polysaccharide Sp1    22

-1

15% EtOAc/PE; [α]D : -10° (c = 1, CHCl3); IR (neat, cm ): 1047, 1147, 1253, 1447, 1508, 1647, 1717, 1795, 2112, 2857; 1H NMR (400 MHz, CDCl3) δ = 0.15 (s, 3H, CH3 TBDMS), 0.16 (s, 3H, CH3 TBDMS), 0.93 (s, 9H, tBu TBDMS), 1.15 (d, 1H, J = 6.3 Hz, H-6), 3.22 (dd, 1H, J = 10.5 Hz, J = 7.7 Hz, H-2), 3.72 (q, J = 6.0 Hz, H-5), 3.88 (dd, 1H, J = 10.5 Hz, J = 3.8 Hz, H-3), 4.15 (dd, 1H, J = 5.1 Hz, J = 2.2 Hz, H-2’), 4.22 (s, 1H, H-4’), 4.25 (d, 1H, J = 3.8 Hz. H-4), 4.40 (d, 1H, J = 11.6 Hz, CHHPh), 4.49 (d, 1H, J = 7.7 Hz, H-1), 4.54 (s, 1H, H-5’), 4.55 (d, 1H, J = 11.9 Hz, CHHPh), 4.58 (d, 1H, J = 11.9 Hz, CHHPh), 4.71 (dd, 1H, J = 5.2 Hz, J = 1.6 Hz, H-3’), 4.86 (d, 1H, J = 11.6 Hz, CHHPh), 5.14 (d, 1H, J = 2.2 Hz, H-1’), 6.82 (d, 1H, J = 9.1 Hz, NH-4), 7.25 – 7.64 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = -5.1 (CH3 TBDMS), -4.5 (CH3 TBDMS), 16.8 (C-6), 18.0 (Cq tBu TBDMS), 25.6 (tBu TBDMS), 51.4 (C-4), 64.5 (C-2), 68.8 (C-5), 71.5 (CH2 Bn), 72.3 (C4’), 74.8 (CH2 Bn), 75.4 (C-2’), 75.6 (C-5’), 76.5 (C-3), 80.3 (C-3’), 92.5 (Cq CCl3), 95.7 (C-1’), 97.2 (C-1), 127.4 – 131.0 (CH Arom), 136.7 (Cq Bn), 137.9 (Cq Bn), 171.9 (C=O); 13C-GATED NMR (100 MHz, CDCl3) δ = 95.7 (J = 157 Hz, C-1’), 97.2 (J = 156Hz, C-1); ESI-MS: 785.2 [M+H]+. O

TCAHN

2-Azido-3-O-(2,4-di-O-benzyl-α-D-galactopyranosylurono-6,3-lactone)-4-trichlor-

acetamido-2,4,6-trideoxy-D-galactopyranose (19): To a stirred solution of 253 mg O O O O BnO compound 18 in 3 mL THF was added 0.09 mL NEt3·3HF (0.6 mmol, 2 equiv.). N3 OH OBn After TLC-analysis indicated complete conversion of starting material the reaction mixture was directly applied on a silica column. Flash chromatography using ethyl acetate/petroleum ether afforded 195 mg of the title compound 19 (0.29 mmol, 99%) as a colorless oil. TLC: 35% EtOAc/PE; IR (neat, cm-1): 696, 729, 817, 906, 1028, 1056, 1109, 1163, 1269, 1508, 1714, 1791, 2113, 2905; α-anomer: 1H NMR (400 MHz, CDCl3) δ = 1.17 (d, 1H, J = 6.3 Hz, H-6), 3.26 (bs 1H, OH-1), 3.41 (dd, 1H, J = 10.6 Hz, J = 3.7 Hz, H-2), 4.16 (dd, 1H, J = 5.2 Hz, J = 2.2 Hz, H-2’), 4.23 (s, 1H, H-4’), 4.33 (dd, 1H, J = 8.9 Hz, J = 3.0 Hz, H-4), 4.40 (d, 1H, J = 11.8 Hz, CHHPh), 4.42 (d, 1H, J = 11.9 Hz, CHHPh), 4.46 (dd, 1H, J = 5.2 Hz, J = 3.6 Hz, H3’), 4.48 (q, 1H, J = 6.4 Hz, H-5), 4.49 (s, 1H, H-5’), 4.68 (dd, 1H, J = 5.2 Hz, J = 1.7 Hz, H-3’), 4.86 (d, 1H, J = 11.8 Hz, CHHPh), 4.87 (d, 1H, J = 11.9 Hz, CHHPh), 5.17 (d, 1H, J = 2.2 Hz, H-1’), 5.31 (d, 1H, J = 3.6 Hz, H-1), 6.79 (d, 1H, J = 9.2 Hz, NH-4), 7.25 – 7.35 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 16.6 (C6), 52.4 (C-4), 59.2 (C-2), 64.2 (C-5), 71.5 (CH2 Bn), 72.2 (C-4’), 74.0 (C-3), 74.8 (CH2 Bn), 75.1 (C-2’), 75.6 (C-5’), 80.3 (C-3’), 92.0 (C-1), 92.6 (Cq CCl3), 95.7 (C-1’), 127.4 – 128.5 (CH Arom), 137.8 (Cq Bn), 137.9 (Cq Bn), 162.7 (C=O TCA), 171.9 (C=O lactone); 13C GATED NMR (100 MHz, CDCl3): δ = 92.0 (J = 172 Hz, C1), 95.7 (J = 161 Hz, H-1’); β-anomer: 1H NMR (400 MHz, CDCl3) δ = 1.28 (d, 1H, J = 6.3 Hz, H-6), 3.32 (dd, 1H, J = 10.4 Hz, J = 8.1 Hz, H-2), 3.78 (q, 1H, J = 6.4 Hz, H-5), 4.00 (dd, 1H, J = 10.3 Hz, J = 3.8 Hz. H-3), 4.01 (bs, 1H, OH-1), 4.16 (dd, 1H, J = 5.2 Hz, J = 2.2 Hz, H-2’), 4.23 (s, 1H, H-4’), 4.27 (dd, 1H, J = 9.1 Hz, J = 3.5 Hz, H-4), 4.51 (s, 1H, H-5’), 4.56 (s, 4H, 2xCH2 Bn), 4.59 (d, 1H, J = 8.0 Hz, H-1), 4.68 (dd, 1H, J = 5.2 Hz, J = 1.7 Hz, H-3’), 5.14 (d, 1H, J = 2.2 Hz, H-1’), 6.86 (d, 1H, J = 9.2 Hz, NH-4), 7.25 – 7.35 (m, 10H, H Arom); C NMR (100 MHz, CDCl3) δ = 16.7 (C-6), 51.4 (C-4), 63.0 (C-2), 69.0 (C-5), 71.5 (CH2 Bn), 72.3 (C-4’), 74.8 (CH2 Bn), 75.2 (C-2’), 75.7 (C-5’), 77.3 (C-3), 80.4 (C-3’), 92.5 (Cq CCl3), 95.8 (C-1’), 96.2 (C-1), 127.4 – 128.5 (CH Arom), 136.7 (2xCq Bn), 162.8 (C=O TCA), 172.0 (C=O lactone); 13C GATED NMR (100 MHz, CDCl3): δ = 95.8 (J = 161 Hz, C-1’), 96.2 (J = 159 Hz, H-1); HRMS: [M+Na]+ calcd for C28H29O9N4Cl3Na

13

693.08923, found 693.08964.

119   

119

Chapter 7    BnO

COOMe O NHTCA

HO BnO

O O N3

O COOMe O

BnO

OMe

OBn

Methyl 2,3-di-O-benzyl-4-O-[2-azido-3-O-(2,4-di-O-benzyl-α-D-galactopyranosyluronate)-4-trichloracetamido2,4,6-trideoxy-D-galactopyranosyl]-β-D-galactopyranoside uronate 2: A solution of 120 mg compound (19) (0.18 mmol), 91 mg diphenylsulfoxide (0.45 mmol, 2.5 equiv.) and 52 mg tri-tert-butylpyrimidine (0.21 mmol, 1.2 equiv.) in 5 mL DCM was stirred over 100 mg activated MS3Å for 30min. The mixture was cooled to -60°C before 35 µL triflic acid anhydride (0.21 mmol, 1.2 eq) was added. The mixture was allowed to warm to -40°C in 1h followed by addition of 121 mg acceptor 5 (0.3 mmol, 1.7 equiv,) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to 0°C. The reaction mixture was then treated with 1 mL pyridine and 0.5 mL Ac2O for 10h followed by addition of 2 mL MeOH. The reaction mixture was diluted with EtOAc and washed subsequently with 2M HCl and sat. NaHCO3 solution. Short filtration over a plug of silica gel afforded the intermediate lactone 20 which was dissolved in MeOH and treated with 2 µL H2SO4 for 12h. The reaction mixture was neutralized upon addition of Et3N followed by concentration under reduced pressure. Flash chromatography (30% EtOAc/hexane) and removal of the eluent afforded the 95 mg of the title compound 2 (0.1 mmol, 58%) as a colorless oil. TLC: 40% EtOAc/PE; [α]D22: +64° (c = 0.6, CHCl3); IR (neat, cm-1): 696, 729, 817, 906, 1028, 1056, 1109, 1163, 1269, 1508, 1714, 1791, 2113, 2905; 1H NMR (500 MHz, CDCl3) δ = 0.91 (d, 1H, J = 6.5 Hz, H-6’), 1.95 (d, 1H, J = 4.0 Hz, OH-3’’), 3.38 – 3.42 (m, 2H, J = 4.0 Hz, J = 3.0 Hz, H-3 and H-2’), 3.58 (s, 3H, CH3 OMe), 3.62 (t, 1H, J = 9.5 Hz, H-2), 3.71 (s, 3H, CH3 COOMe), 3.81 (s, 3H, CH3 COOMe), 3.90 (dd, 1H, J = 10.0 Hz, J = 3.5 Hz, H-2’’), 3.96 (s, 1H, H-5), 4.15 – 4.20 (m, 2H, H-3’ and H-3’’), 4.21 (s, 1H, J = 8.0 Hz, H-1), 4.27 (s, 1H, H-4’’), 4.36 (s, 1H, H-4), 4.49 (d, 1H, J = 12.5 Hz, CHHPh), 4.50 (s, 1H, H-5’), 4.51 (s, 1H, H-4’), 4.54 (d, 1H, J = 11.5 Hz, CHHPh), 4.58 (s, 1H, H-5’’), 4.70 (d, 1H, J = 11.5 Hz, CHHPh), 4.74 (d, 1H, J = 11.0 Hz, CHHPh), 4.76 (s, 1H, CH2 Bn), 4.85 (d, 1H, J = 13.0 Hz, CHHPh), 4.90 (d, 1H, J = 11.0 Hz, CHHPh), 5.05 (d, 1H, J = 3.5 Hz, H-1’), 5.61 (d, 1H, J = 2.5 Hz, H-1’’), 6.70 (d, 1H, J = 10.0 Hz, NH-4’), 7.16 – 7.46 (m, 20H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 16.1 (C-6’), 51.4 (C-4’), 52.2 (CH3 COOMe), 52.6 (CH3 COOMe), 57.5 CH3 OMe), 60.1 (C-2’), 65.6 (C-5’), 69.2 (C-3’’), 70.1 (C-3’), 70.9 (C-5’’), 72.7 (CH2 Bn), 73.4 (CH2 Bn and C-5), 74.6 (C-2’’), 74.9 (CH2 Bn), 75.2 (CH2 Bn), 76.2 (C-4), 77.8 (C4’’), 78.1 (C-2), 78.8 (C-3), 92.7 (Cq CCl3), 92.9 (C-1’’), 97.1 (C-1’), 105.1 (C-1), 127.6 – 131.0 (CH Arom), 137.9 (Cq Bn), 138.0 (Cq Bn), 138.2 (Cq Bn), 138.3 (Cq Bn), 162.6 (C=O TCA), 168.4 (C=O COOMe), 168.7 (C=O COOMe); 13C GATED NMR (150 MHz, CDCl3): δ = 92.9 (J = 171 Hz, C-1’’), 97.1 (J = 168 Hz, C-1’), 105.1 (J = 159 Hz, C-1); HRMS: [M+H]+ calcd for C51H58O16N4Cl3 1086.28351, found 1087.29438.

References and Notes 1 2

Weintraub, A. Carbohydr. Res. 2003, 338, 2539–2547. Harding, C.V.R.W.; Allen, R.P.M.; Unanue, E.R. Proc. Natl. Ac. Sci. USA 1991, 88, 2740–2744.

3

(a) Tzianabos, A.O.; Wang, J.Y.; Lee, J.C. Proc. Natl. Ac. Sci. USA 2001, 98, 9365–9370 (b) Tzianabos, A.O.; Chandrakar, A.; Kalka-Moll, W.; Stingele, F.; Dong, V.M.; Finberg, R.W.; Peach, R.; Sayegh, M.H. Infect. Immun. 2000, 68, 6650–6655 (c) Kalka-Moll, W.M.; Tzianabos, A.O.; Bryant, P.W.; Niemeyer, M.; Ploegh, H.L.; Kasper, D.L. J. Immunol. 2002, 169, 6149–6153 (d) Cobb, B.A.; Wang, Q.; Tzianabos, A.O.; Kasper, D.L. Cell 2004, 117, 677–687.

4

Tzianabos, A.O.; Wang, J.Y.; Kasper, D.L. Carbohydr. Res. 2003, 338, 2531-2538.

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6 7 8

(a) How, M.J.; Brimacombe, J.S.; Stacey, M. Adv. Carbohydr. Chem. Biochem. 1964, 19, 303–358 (b) Guy, R.C.E.; How, M.J.; Stacey, M.; Heidelberger, M. J. Biol. Chem. 1967, 242, 5106–5111 (c) Lindberg, B.; Lindqvist, B.; Lönngren, J.; Powell, D.A. Carbohydr. Res. 1980, 78, 111–117. Tzianabos, A.O.; Onderdonk, A.B.; Rosner, B.; Cisneros, R.L.; Kasper, D.L Science 1993, 262, 416–419. Choi, Y.-H.; Roehrl, M.H.; Kasper, D.L.; Wang, J.Y. Biochemistry 2002, 41, 15144–15151. (a) Garcia, B.A.; Poole, J.L.; Gin, D.Y. J. Am. Chem. Soc. 1997, 119, 7597–7598 (b) Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279.

9

Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522. 10 (a) See Chapter 2 (b) Van den Bos, L.J.; Codée, J.D.C.; Van der Toorn, J.C.; Boltje, T.J.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2004, 6, 2165–2168. 11 (a) See Chapter 6 (b) Van den Bos, L.J.; Codée, J.D.C.; Van Boom, J.H.; Overkleeft, H.S.; Van der Marel, G.A. Org. Biomol. Chem. 2003, 1, 4160–4165. 12 Lönn, H.; Lönngren, J. Carbohydr. Res. 1984, 132, 39–44. 13 Compain, M.; Mesrari, L.; Anker, D.; Doutheau, A. Carbohydr. Res. 1999, 316, 201–205. 14 Boyer, V.; Stanchev, M.; Fairbanks, A.J.; Davis, B.G. Chem. Commun. 2001, 19, 1908–1909. 15 This reaction exploits the slight reactivity difference between all four hydroxyl substituents at the carbohydrate core. Du, Y.; Zhang, M.; Kong, F. Org. Lett. 2000, 2, 3797–3800. 16 The corresponding 1-thio-α-D-mannopyranoside did show silyl migration. See Chapter 4 (compound 9). 17 (a) See Chapter 3 (b) Van den Bos, L.J.; Litjens, R.E.J.N.; Van den Berg, R.J.B.H.N.; Overkleeft, H.S.; Van der Marel, G.A. Org. Lett. 2005, 7, 2007–2010. 18 Hermans, J.P.G.; Elie, C.J.J.; Van der Marel, G.A.; Van Boom, J.H. J. Carbohydr. Chem. 1987, 6, 451–462. 19 (a) Seeberger, P.H.; Baumann, M.; Zhang G.; Kanemitsu, T.; Swayze, E.E.; Hofstadler, S.A.; Griffey, R.H. Synlett 2003, 9, 1323–1326 (b) Liang, F.-S.; Wang, S.-K.; Nakatani, T.; Wong, C.-H. Angew. Chem. Int. Ed. 2004, 43, 6496–6500. 20 (a) Liav, A.; Jacobson, I.; Sheinblatt, M.; Sharon, N. Carbohydr. Res. 1978, 66, 95–101 (b) Hasegawa, A.; Tanakashi, E.; Goh, Y.; Kiso, M. Carbohydr. Res. 1982, 103, 263–272 (c) Liang, H.; Grindley, T.B. J. Carbohydr. Chem. 2004, 23, 71–82. 21 Medgyes, A.; Farkas, E.; Lipták, A.; Pozsgay, V. Tetrahedron 1997, 53, 4159–4178. 22 Kinzy, W.; Schmidt, R.R. Liebigs Ann. Chem. 1985, 8, 1537–1545. 23 (a) Smid, P.; Jörning, W.P.A.; Van Duuren, A.M.G.; Boons, G.-J.P.H.; Van der Marel, G.A.; Van Boom, J.H. J. Carbohydr. Chem. 1992, 11, 849–865. 24 This result again confirms the highly α-selective nature of this donor. See Chapters 3 and 9. 25 The regular trimethylphosphine reagent can not be used for the reduction of the azide functionality in the presence of a trichloroacetamide group. Attempts using more sterically hindered phosphines (otolylphosphine) showed no reaction. Faul, D.; Himbert, G. Liebigs Ann. Chem. 1986, 8, 1466–1473. 26 Haller, M.; Boons, G.-J. J. Chem. Soc., Perkin Trans. 1 2001, 814–822. 27 Crich, D.; Smith, M.; Yao, Q.; Picione, J. Synthesis 2001, 323–326. 28 Fales, H.M.; Jaouni, T.M.; Babashak, J.F. Anal. Chem. 1973, 45, 2302–2303.

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Chapter 8 │  Synthetic Study Towards               the PSA1 Tetrasaccharide      Repeating Unit

Abstract: A synthetic study towards the protected tetrasaccharide repeating unit of zwitterionic polysaccharide PSA1 using 1-thio, 1-seleno and 1-hydroxyl functionalized donor glycosides is presented. The ABC trisaccharide part was successfully assembled using an iterative dehydrative glycosylation protocol.1

Introduction Zwitterionic polysaccharide A1 (PSA1), isolated from capsules of the anaerobic bacterium Bacteriodes fragilis, is thought to be the key pathogenic substance responsible for the development of intraabdominal sepsis.2,3 In contrast to what is found for the majority of pathogenic polysaccharides,4 the immune response elicited by PSA1 is mediated by Tlymphocytes.5 This unusual immunologic property of PSA1 is likely caused by its zwitterionic character. Neutralization of either the positively charged amino groups or the negatively charged carboxyl groups resulted in a strongly reduced biological activity as compared to the unmodified polysaccharide construct.6,7 The repeating unit of PSA1 (1) is depicted in Figure 1. The positive charge is situated at the amino group of the rare 2acetamido-4-amino fucose moiety and the negative charge resides in the carboxylate of the pyruvate ketal spanning C4 and C6 in the galactopyranosyl residue.

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Retrosynthetic analysis indicates that the protected tetrasaccharide repeating unit 2 can be assembled from protected monosaccharides 3-8 following the sequence of glycosylation events depicted in Figure 1. The lengthy synthetic route to suitably protected 2,4-diamino 2,4,6-trideoxygalactose building blocks 7 or 8 dictates that this residue is best introduced at a late stage (i.e. the third glycosylation in Figure 1). The construction of the protected trisaccharide (ABC) that follows from this reasoning was investigated using orthogonal and chemoselective glycosylation strategies in combination with Ph2SO/Tf2O as activator system.8 The orthogonal strategy9 required the availability of furanoside 3,10 1-seleno galactosazide 411 and terminal pyruvate residue 6, while for the chemoselective strategy12 the availability of furanoside 3, diol acceptor 513 and pyruvate building block 6 was necessary. Figure 1 BzO TCAHN

H3N O AcHN

HO

O

O

1st glycosylation

OH

BzO H

O O AcHN

HO

D

AcO

O HO H

3rd glycosylation

OH

O A

O

2nd glycosylation OBn

O

O O O

BzO

N3

OH Ph

BzO

3

C

MeOOC

O O

O

O HO 4: R = α-SePh N3 5: R = α/β-OH

MeO2C R2HN

O

O O

HO

O AcO

R1

N3 1

R

Me

OiPr

Me 2

O O

OBz

OBz O

OBz

Me 1

O

O

B

O

OH OOC

BzO

O N3

H

BzO

6

OiPr

OBz

2

7: R = β-SPh, R = Cbz 8: R1 = α/β-OH, R2 = TCA

Results and Discussion Pyruvate derivative 6 was synthesized as depicted in Scheme 1. Acidolysis of the 4,6-Obenzylidene in known thiogalactopyranoside 914 gave diol 10. The pyruvate ketal was introduced according to the procedure developed by Ziegler and coworkers.15 Treatment of 10 with equimolar amounts of methyl pyruvate and borontrifluoride diethyletherate (BF3·OEt2) in acetonitrile gave pyruvated galactopyranoside 11. Conclusive evidence for the predicted (R)-stereochemistry at the pyruvate ketal came from the X-ray structure of 11 (Scheme 1). The iso-propyl group was installed at the anomeric centre via a Ph2SO/Tf2O-mediated glycosylation to give 12. Although the use of tri-tert-butylpyrimidine (TTBP)16 in this condensation was omitted to prevent ortho-ester formation, no deterioration of the putative acid labile pyruvate acetal was observed. In the last step, hydrogenolysis of the C3 benzyl protective group yielded galactopyranoside 6.

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Zwitterionic Polysaccharide PSA1    Scheme 1. Me R2O

OR1 O

BnO

O O

MeOOC b SPh

O BnO

OBz a

X-ray crystal structure of pyruvate derivative 11

SPh

OBz 11

9: R1=R2=CHPh 10: R1=R2=H

c Me O O

MeOOC

O RO

O

d

OBz 12: R = Bn 6: R = H

Reagents and Conditions: (a) TsOH, MeOH, 97% (b) methyl pyruvate, BF3·OEt2, ACN, 74% (c) Ph2SO, Tf2O, -60°C then iso-propanol, 77% (d) H2, Pd/C, MeOH, 81%.

The synthesis of trisaccharide 15 was investigated employing first an orthogonal glycosylation approach, in which a hemiacetal donor is condensed with a 1-seleno acceptor (Scheme 2A).9 Pre-activation of galactofuranoside 310 using the Ph2SO/Tf2O17,18 reagent combination at -40°C and subsequent addition of 1.5 equivalents of partially protected 1selenogalactoside 4 afforded β-linked digalactoside 13 in a yield of 56%.9 Although the anomeric seleno-function was stable according to TLC-analysis, the yield of this condensation could not be further improved. In the next glycosylation event, 1-selenodisaccharide 13 was successfully activated with the Ph2SO/Tf2O reagent combination. However, addition of acceptor 6 did not afford the expected trisaccharide 15. The same outcome was observed employing the BSP/Tf2O activator system.19 Previous experiences have shown that NIS/TMSOTf is a useful alternative when sulfonium ion-mediated glycosidation of thioglycosides is unproductive.20 Indeed, when a mixture of disaccharide 13 and acceptor 6 was treated with the NIS/TMSOTf 21 in DCM at 0°C trisaccharide 15 was obtained in 65% yield as the single stereoisomer.22 Most likely, the use of only a catalytic amount of TMSOTf is at the basis of this favorable result.20 With the objective to assemble trimer 15 via a one-pot glycosylation procedure, the chemoselective dehydrative glycosylation strategy (Scheme 2A) was investigated.12 Preactivation of 1-hydroxyl donor 3 with Ph2SO/Tf2O for 1h at -40°C was followed by addition of a solution of diol 5 in DCM/1,4-dioxane23 (10/1) to give, upon warming of the reaction mixture, disaccharide 14 in 60% yield. Analogously, Ph2SO/Tf2O-mediated dehydrative coupling of dimer 14 with acceptor 6 afforded target trisaccharide 15 in 52% yield. Next, attention was focused on adapting these results in a one-pot iterative dehydrative glycosylation strategy (Scheme 2B). Pre-activation of donor 3 and addition of acceptor 5

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afforded hemiacetal donor 14 with concomitant regeneration of Ph2SO. The reaction mixture was warmed to room temperature, kept overnight and then cooled down to -60°C. Activation of the newly formed hemiacetal disaccharide 14 was accomplished with the addition of a fresh amount of Tf2O and stirring was continued for 1h at -40°C. Addition of 1.5 equivalent of acceptor 6 and TTBP followed by overnight warming to room temperature produced trisaccharide 15 in 62% yield. Removal of all the protective groups in 15 was accomplished by: 1) conversion of the azide functionality into the acetamido group, 2) acidic cleavage of the 4,6-O-benzylidene acetal, and 3) alkaline hydrolysis of all ester functions gave unprotected trisaccharide 16 in 50% yield (Scheme 2).

Scheme 2. A: sequential approaches BzO

H

O

O

OH

BzO

+ BzO

Ph

Ph

O O

O BzO O

HO

a or b

3

O

O

O N3

N3 R 4: R=α-SePh 5: R=α/β-OH

OBz

H

BzO BzO

OBz

R

13: R=α-SePh 14: R=α/β-OH c or d

B: one-pot approach Ph2SO Tf2O TTBP 3

R1O Tf2O

5

1h -60°C

16h -40°C

6 TTBP

1h rT -60°C

16h -40°C rT

R2O H

R1O

OR2 O

O

OR1

R3 O R1O

62%

O

O

OR1 R4OOC

O

O

O

Me e

15: R1=Bz, R2=benzylidene, R3=N3, R4=Me 16: R1=R2=R4=H, R3=NHAc

Reagents and conditions: (a) 3, Ph2SO, Tf2O, TTBP, -40°C, DCM then 4, 56% (b) 3, Ph2SO, Tf2O, TTBP, -40°C, DCM/dioxane (10/1) then 5, 60% (c) 13, 6, NIS, TMSOTf, DCM, 65% (d) 14, Ph2SO, Tf2O, TTBP, -40°C, DCM then 6, 52% (e) 1) 1M Me3P in toluene, THF/H2O (4/1), 2) Ac2O, pyridine, 3) TsOH, MeOH, 4) KOH, THF/H2O, 50% (over 4 steps).

Now the stage was set to study the construction of the fully protected tetrameric repeating unit. First, the glycosylation of trisaccharide 26 with donor 7 was investigated (Scheme 4). Synthesis of compound 7 started from known key intermediate 17,24 which was obtained after a lengthy route from commercially available glucosamine hydrochloride (Scheme 3.).25 Following a classical sequence of reactions, C6 de-oxygenated derivative 20 was obtained. First, cleavage of the benzylidene function (18) followed by regioselective tosylation and iodination under Finkelstein conditions (19) and finally mild reduction of the C6 iodide using

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Zwitterionic Polysaccharide PSA1   

Zn/AcOH gave quinovosamine derivative 20 in good overall yield. Inversion at OH4 was accomplished by first triflation under standard conditions followed by treatment with sodium azide giving orthogonally functionalized 21 in 88%. The last part of the synthesis consisted of amine protective group interchanges in order to introduce an azide at the C2 position, which should ensure α-selectivity during glycosylation. The 3-O-acetyl group was first exchanged for a 3-O-tert-butyldimethylsilyl group (22) preventing an intra-molecular aza-Wittig reaction during azide reduction. Treatment of compound 22 with trimethylphosphine (Me3P) in a THF/water mixture and subsequent re-protection of the free amine as a Cbz-group gave compound 23 in 94% yield. One-pot removal of the N-phthaloyl protection using ethylenediamine and diazotransfer on the liberated amine function afforded compound 24 in 84% yield. Exchange of the acid labile C3 silyl protection for an acetyl delivered target compound 7 in a yield of 90%.

Scheme 3. 1

R2O AcO

OR O

b SPh

R HO AcO

NPhth a

17: R1=R2=benzylidene 18: R1=R2=H

c

CbzHN

O

d SPh

O RO

NPhth 19: R = I 20: R = H

SPh

NPhth 21: R = Ac 22: R = TBS

e

CbzHN g

O RO

SPh

O TBSO

24: R = TBS 7: R = Ac

f

SPh NPhth

N3 h

N3

23

Reagents and conditions: (a) 80% AcOH, H2O, 80°C, 95% (b) 1) TsCl, pyridine, 2) NaI, 2-butanone, reflux, 88% (over 2 steps) (c) Zn, AcOH, DCM, 73% (d) 1) Tf2O, pyridine, 0°C, 2) NaN3, DMF, 88% (e) 1) KOtBu, MeOH, 2) TBDMSCl, imidazole, 81% (f) 1) 1M Me3P in toluene, toluene/H2O (100/5), 2) CbzOSu, Et3N, DCM, 94% (over 2 steps) (g) 1) (CH2NH2)2, n-BuOH, 2) TfN3, K2CO3, CuSO4, DCM/H2O/MeOH, 84% (over 2 steps) (h) 1) TBAF, THF, 2) Ac2O, pyridine, 90% (over 2 steps).

Compound 26 was obtained after reductive opening of the benzylidene functionality in compound 15 using the triethylsilane/triflic acid reagent combination (Scheme 4).26 Although activation of thio donor 7 with Ph2SO/Tf2O proceeded smoothly at -60°C, addition of trisaccharide acceptor 17 did not lead to a productive coupling. Changing the activator to the NIS/TMSOTf system was also unsuccessful because donor 7 could not be activated under these conditions. Next, attention turned to Gin’s dehydrative glycosylation procedure using donor 8 obtained in a straightforward manner from compound 3027 (Scheme 4). Activation of hemiacetal donor 8 using the Ph2SO/Tf2O reagent combination and addition of trisaccharide 17 afforded tetrasaccharide 2 in 17% yield. Trisaccharide 17 was recovered in 53%, whereas excess donor 8 completely degraded to unidentified baseline products.

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Chapter 8    Scheme 4. RHN R2O BzO BzO

R3HN R2 O

O N3

a

H

+ R

O

OR1 O

O

BzO

7: R1=β-SPh, R2=Ac, R3=Cbz 25: R1=β-OTBS, R2=H, R3=TCA 8: R1=α/β-OH, R2=Ac, R3=TCA

O

OBz MeOOC

N3

OBz

N3 O

1

O

R2O

O O

O

BzO

c

H

O

OBn O

O

O

BzO BzO

15: R1=R2=-benzylidene 26: R1=Bn, R2=H

O

OBz

Me b

OBz

N3 O

27: R=Cbz 2: R=TCA

MeOOC

O

O

O

Me

Reagents and conditions: (a) 1) Ac2O, pyridine, 2) TBAF, AcOH, THF, 77% (over 2 steps) (b) Et3SiH, TfOH, DCM, -50°C, 92% (c) 8, Ph2SO, Tf2O, TTBP (1 equiv. wrt donor 8), DCM, -40°C then 26, 17% (53% recovered acceptor 26).

Conclusion This Chapter describes the synthesis of the protected tetrasaccharide corresponding to the repeating unit of the zwitterionic polysaccharide PSA1. Current efforts are directed to overcoming the poor yield in the final glycosylation step. Possibly, the difficulty in introducing the 2,4-diaminofucose at this stage results from a combination of the steric bias in the trimeric acceptor 26 and the intrinsic low reactivity of the protected 2,4-diaminofucose donors 7 and 8. On the positive side, comparative studies towards the construction of the protected trisaccharide 15 demonstrated the effectiveness of the dehydrative glycosylation procedure and led to the development of a one-pot iterative protocol that is characterized by the single use of the Ph2SO/Tf2O reagent combination.

Experimental Section General Procedures: 1H and 13C NMR spectra were recorded on a Jeol JNM-FX-200 (200/50.1 MHz), a Brüker AV-400 (400/100 MHz) and a Brüker DMX-600 (600/150 MHz) spectrometer. Chemical shifts are given in ppm (δ) relative to tetramethylsilane as internal standard. Coupling constants are given in Hz. All given 13C spectra are proton decoupled. Mass spectra were recorded with PE/SCIEX API 165 with electronspray interface and QStar Applied Biosystems Q-TOF (TOF-section). Optical rotations were measured on a Propol automatic polarimeter. IR-spectra were recorded on a Shimadzu FTIR-8300. Melting points were measured on a Büchi Schmelzpunktbestimmungsapparat nach dr. Tottoli (+ pat 320.388) and were uncorrected. Traces of water in the donor and acceptor glycosides, diphenylsulfoxide, benzenesulfinylpiperidine and TTBP where removed by coevaporation with toluene. Benzenesulfinylpiperidine19 and TTBP16 were synthesized as described by Crich et al. Dichloromethane (DCM, Baker p.a.) was boiled under reflux over P2O5 for 2h and distilled immediately prior to use. Trifluoromethanesulfonic anhydride was distilled from P2O5. Molecular sieves 3Å were flame dried before use. Solvents used for flash chromatography and TLC were of technical grade and distilled before use. Flash chromatography was performed on Baker silica gel (0.063 – 0.200 mm). TLC-analysis was conducted on DC-

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Zwitterionic Polysaccharide PSA1    fertigfolien (Schleicher & Schuell, F1500, LS254) with detection by UV-absorption (254 nm) where applicable and by spraying with 20% sulfuric acid in ethanol followed by charring at ~150°C or by spraying with a solution of (NH4)6Mo7O24·H2O (25 g/L) and (NH4)4Ce(SO4)4·2H2O (10g/L) in 10% sulfuric acid in water followed by charring at ~150°C. Phenyl 2-O-benzoyl-3-O-benzyl-1-thio-β-D-galactopyranoside (10): Known compound 928 (100 mg, 0.18 mmol) was suspended in 5 mL MeOH and cat. TsOH was added. The BnO SPh OBz reaction mixture was stirred until TLC indicated complete conversion of starting material into a lower running product. The reaction mixture was neutralized by the addition of 0.5 mL NEt3 and HO

OH O

concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 81 mg of the title compound 15 (0.18 mmol, quant.) as a colorless oil. TLC: 30% EtOAc/PE; [α]D22: +41° (c = 1, CHCl3); IR (neat, cm-1): 694, 995, 1056, 1257, 1720, 2923; 1H NMR (200 MHz, CDCl3) δ = 3.16 (bs, 2H, OH-4, OH-6), 3.67 – 3.73 (m, 2H, H-3, H-5), 3.84 – 4.06 (m, 2H, H-6), 4.22 (d, 1H, J = 2.6 Hz, H-4), 4.65 (d, 1H, J = 12.0 Hz, CHHPh), 4.71 (d, 1H, J = 12.0 Hz, CHHPh), 4.78 (d, 1H, J = 10.2 Hz, H-1), 5.56 (t, 1H, J = 9.5 Hz, H2), 7.15 – 8.05 (m, 15H, H Arom); 13C NMR (50 MHz, CDCl3) δ = 62.0 (C-6), 66.3, 69.6, 78.4, 79.2 (C-2, C-3, C-4, C-5) 71.1 (CH2 Bn), 86.5 (C-1) 127.6 – 133.1 (CH Arom), 134.1 (Cq, SPh), 136.9 (Cq Bn) 165.3 (C=O Bz); HRMS: [M+Na]+ calcd for C26H26O6SNa 489.13423, found 489.13465. Me

O

BnO

Phenyl 2-O-benzoyl-3-O-benzyl-4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-1-thio-β(11): To a solution of 4.66 g compound 10 (10 mmol) in 50 mL ACN were added 1.84 mL methylpyruvate (20 mmol, 2.0 equiv.) and 2.50 mL BF3·Et2O (20 mmol, 2.0 equiv.). The reaction mixture was stirred for 3h at ambient temperature under Ar-atmosphere, followed by the addition of 1 mL Et3N and 250 mL EtOAc.

D-galactopyranoside

O O

MeOOC

SPh

OBz

Subsequently, the mixture was washed with sat. aq. NaHCO3-solution (3x150 mL). The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 3.43 g of compound 11 (7.4 mmol, 74%) as a yellow oil. To determine the crystal structure, the product 11 was recrystallized from boiling ethanol. TLC: 30% EtOAc/PE; [α]D22: +1° (c = 1, CHCl3); IR (neat, cm-1): 694, 748, 979, 1072, 1203, 1257, 1458, 1519, 1712; 1H NMR (400 MHz, CDCl3) δ = 1.58 (s, 3H, CH3 Me), 3.39 (s, 1H, H-5), 3.67 (dd, 1H, J = 9.2 Hz, J = 3.2 Hz, H-3), 3.81 (s, 3H, Me COOMe), 3.92 (d, 1H, J = 12.8 Hz, H-6), 4.09 (d, 1H, J = 12.8 Hz, H-6), 4.37 (s, 1H, H-4), 4.55 (d, 1H, J = 12.8 Hz, CHHPh), 4.75 (d, 2H, J = 11.6 Hz, H-1, CHHPh), 5.53 (t, 1H, J = 9.6 Hz, H-2), 7.13 – 8.05 (m, 15H, H Arom); 13 C NMR (100 MHz, CDCl3) δ = 25.4 (CH3 Me), 52.2 (CH3 COOMe), 65.3 (C-6), 67.6 (C-4), 68.5 (C-2), 68.7 (C-3), 69.4 (CH2 Bn), 76.9 (C-5), 85.4 (C-1), 98.3 (Cq pyruvate), 127.0 – 132.9 (CH Arom), 129.9 (Cq SPh), 131.7 (Cq Bz), 137.2 (Cq Bn), 164.7 (C=O COOMe), 170.2 (C=O Bz); HRMS: [M+Na]+ calcd for C30H30O8SNa 573.15536, found 573.15443. Isopropyl 2-O-benzoyl-3-O-benzyl-4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-1thio-β-D-galactopyranoside (12): A solution of 2.20 g donor 11 (1 equiv.), 1.2 g

Me MeOOC

O O

Ph2SO (5.8 mmol, 1.5 equiv.) in 60 mL DCM was stirred over activated MS3Å for 30min. The mixture was cooled to -60°C before 0.9 mL triflic acid anhydride (5.4 OBz mmol, 1.4 equiv.) was added. The mixture was allowed to warm to -40°C over 15min at which time 3.82 mL iso-propanol (50 mmol, 13 equiv.) was added. Stirring was continued and the reaction BnO

O

O

mixture was allowed to warm to -20°C. The reaction mixture was neutralized with 2.5 mL NEt3, diluted with EtOAc and washed with water. The aqueous phase was extracted twice with 100 mL EtOAc. The combined organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography

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Chapter 8    (EtOAc/PE) and removal of the eluent afforded 1.54 g of the title compound 12 (3.1 mmol, 77%) as a white solid. TLC: 20% EtOAc/PE; [α]D22: +10° (c = 1, CHCl3); IR (neat, cm-1): 694, 748, 979, 1072, 1203, 1257, 1458, 1519, 1712; 1H NMR (400 MHz, CDCl3) δ = 1.00 (d, 1H, J = 6.2 Hz, CH3 isoprop), 1.18 (d, 1H, J = 6.2 Hz, CH3 isoprop), 1.67 (s, 3H, CH3 Me), 3.30 (s, 1H, H-5), 3.62 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz, H-3), 3.86 (s, 3H, CH3 COOMe), 3.89 (m, 1H, J = 6.2 Hz, CH isoprop), 3.97 (dd, 1H, J = 12.6 Hz, J = 1.6 Hz, H-6), 4.10 (dd, 1H, J = 12.6 Hz, J = 1.6 Hz, H-6), 4.32 (d, 1H, J = 3.6 Hz, H-4), 4.54 (d, 1H, J = 8.0 Hz, H-1), 4.58 (d, 1H, J = 12.8 Hz, CHHPh), 4.80 (d, 1H, J = 12.8 Hz, CHHPh), 5.49 (dd, 1H, J = 10.0 Hz, J = 8.0 Hz, H-2), 7.18 – 8.05 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 21.9 (CH3 isoprop), 23.2 (CH3 isoprop), 25.8 (CH3 Me), 52.5 (CH3 COOMe), 65.5 (C-6), 65.7 (C-5), 67.8 (C-4), 69.5 (CH2 Bn), 70.8 (C-2), 72.2 (CH isoprop), 75.9 (C-3), 98.7 (Cq pyruvate) 99.9 (C-1), 127.5 – 132.7 (CH Arom), 130.4 (Cq Bz), 137.6 (Cq Bn), 165.0 (C=O COOMe), 170.7 (C=O Bz); HRMS: [M+Na]+ calcd for C27H32O9Na 523.19385, found 523.19360. Isopropyl 2-O-benzoyl-4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-1-thio-β-Dgalactopyranoside (6): To a solution of 3.09 g compound 12 (5.47 mmol) in 20 mL

Me O O

MeOOC

MeOH was added 0.61 g of Pd/C (10%). The mixture was stirred for 12h at ambient temperature under an atmosphere of H2 gas followed by filtration over Hyflo and OBz concentration under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 1.65 g of compound 6 (4.0 mmol, 81%) as a white solid. TLC: 40% EtOAc/PE; O

HO

O

[α]D22: +39° (c = 1, CHCl3); IR (neat, cm-1): 694, 748, 979, 1072, 1203, 1257, 1458, 1519, 1712; 1H NMR (400 MHz, CDCl3) δ = 1.06 (d, 1H, J = 6.4 Hz, CH3 isoprop), 1.20 (d, 1H, J = 6.4 Hz, CH3 isoprop), 1.64 (s, 3H, CH3 Me), 2.73 (bs, 1H, OH-3), 3.30 (d, 1H, J = 1.2 Hz, H-5), 3.82 (s, 3H, CH3 COOMe), 3.62 (dd, 1H, J = 10.0 Hz, J = 4.0 Hz, H-3), 3.89 (m, 1H, J = 6.2 Hz, CH isoprop), 3.97 (dd, 1H, J = 12.6 Hz, J = 1.6 Hz, H-6), 4.10 (dd, 1H, J = 12.6 Hz, J = 1.6 Hz, H-6), 4.32 (d, 1H, J = 3.6 Hz, H-4), 4.54 (d, 1H, J = 8.0 Hz, H-1), 4.58 (d, 1H, J = 12.8 Hz, CHHPh), 4.80 (d, 1H, J = 12.8 Hz, CHHPh), 5.49 (dd, 1H, J = 10.0 Hz, J = 8.0 Hz, H-2), 7.18 – 8.05 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 21.9 (CH3 isoprop), 23.2 (CH3 isoprop), 25.8 (CH3 Me), 52.5 (CH3 COOMe), 65.5 (C-6), 65.7 (C-5), 67.8 (C-4), 69.5 (CH2 Bn), 70.8 (C-2), 72.2 (CH isoprop), 75.9 (C-3), 98.7 (Cq pyruvate) 99.9 (C-1), 127.5 – 132.7 (CH Arom), 130.4 (Cq Bz), 137.6 (Cq Bn), 165.0 (C=O COOMe), 170.7 (C=O Bz); HRMS: [M+Na]+ calcd for C20H26O9Na 433.14690, found 433.14559. Phenyl 3-O-acetyl-2-phthalimido-2-deoxy-1-thio-β-D-glucopyranoside (18): Known compound 1724 (100 mg, 0.19 mmol) was suspended in 5 mL MeOH and cat. TsOH was NPhth added. The reaction mixture was stirred until TLC indicated complete conversion of starting material into a lower running product. The reaction mixture was neutralized by the addition of 0.5 mL NEt3 and HO AcO

OH O

SPh

concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 83 mg of the title compound 18 (0.19 mmol, quant.) as a colorless oil. TLC: 20% EtOAc/PE; [α]D22: +20° (c = 1, CHCl3); IR (neat, cm-1): 709, 1110, 1257, 1380, 1712; 1H NMR (200 MHz, CDCl3) δ = 1.91 (s, 3H, CH3 Ac), 2.23 (s, 1H, OH-6), 3.06 (s, 1H, OH-4), 3.66 – 4.01 (m, 4H, H-4, H-5, H-6, H-6’), 4.29 (t, 1H, J = 10.2 Hz, H-2), 4.34 (d, 1H, J = 10.6 Hz, H-1), 5.68 (t, 1H, J = 8.8 Hz, H-3), 7.24 – 7.91 (m, 9H, H Arom); 13C NMR (50 MHz, CDCl3) δ = 20.6 (CH3 Ac), 53.7 (C-2), 62.3 (C-6), 69.6, 74.4, 79.6 (C-3, C-4, C-5), 83.0 (C-1), 123.6 – 134.2 (CH Arom), 131.4 (Cq SPh), 167.1 (C=O NPhth), 167.9 (C=O NPhth), 171.2 (C=O Ac); HRMS: [M+Na]+ calcd for C22H21O7NSNa 466.09309, found 466.09249. I HO AcO

O

SPh

NPhth

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130

Phenyl 3-O-acetyl-6-iodo-2-phthalimido-2,6-dideoxy-1-thio-β-D-glucopyranoside (19): A solution of 44.3 g compound 18 (100 mmol) in 500 mL anhydrous pyridine was cooled to 0˚C followed by the addition of 21 g TsCl (110 mmol, 1.1 equiv.). The reaction mixture

Zwitterionic Polysaccharide PSA1    was stirred for 12h under Ar-atmosphere at 0˚C. The reaction was quenched by addition of ice cold MeOH. The mixture was diluted with 1 L EtOAc and washed twice with 400 mL 1M HCl solution and 400mL NaHCO3 (sat. aq.). The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The crude oil was dissolved in 500 mL 2-butanone and 21 g NaI (140 mmol, 1.4 equiv.) was added. The mixture was refluxed for 4 days at 90˚C. The reaction mixture was cooled to ambient temperature, diluted with 1 L EtOAc and washed twice with 400 mL 1M Na2S2O3 and 300 mL H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether yielded 49.0 g compound 19 (88 mmol, 88%) as a yellow solid. TLC: 20% EtOAc/PE; IR (neat, cm-1): 709, 1110, 1257, 1380, 1712; 1H NMR (200 MHz, CDCl3) δ = 1.89 (s, 3H, CH3 Ac), 3.03 (d, 1H, J = 5.4 Hz, OH-4), 3.39 – 3.69 (m, 4H, H-4, H-5, 2xH-6), 4.27 (t, 1H, J = 10.2 Hz, H-2), 5.70 (t, 1H, J = 8.4 Hz, H-3), 5.79 (d, 1H, J = 10.6 Hz, H-1), 7.23 – 7.77 (m, 9H H Arom); 13C NMR (50 MHz, CDCl3) δ = 6.0 (C-6), 20.6 (CH3 Ac), 53.5, 73.6, 74.1, 78.3, 82.6, 86.7 (C-1, C-2, C-3, C-4, C-5), 123.6 – 134.4 (CH Arom), 130.7 (Cq NPhth), 130.9 (Cq NPhth), 167.0 (C=O Nphth), 167.8 (C=O NPhth), 171.3 (C=O Ac); HRMS: [M+Na]+ calcd for C22H20O6NISNa 575.99482, found 575.99487.

HO AcO

O

SPh

NPhth

Phenyl 3-O-acetyl-2-phthalimido-2,6-dideoxy-1-thio-β-D-glucopyranoside (20): To a solution of 49 g compound 19 (88 mmol) in 440 mL DCM and 43 mL AcOH (740 mmol, 8.4 equiv.) was added 127 g Zn-powder (1936 mmol, 22 equiv.). The mixture was stirred

for 6h at ambient temperature under Ar-atmosphere. The reaction mixture was filtered and concentrated under reduced pressure. Flash column chromatography using ethylacetate/petroleum ether afforded 27.4 g of compound 20 (64 mmol, 73%) as a white solid. TLC: 30% EtOAc/PE; [α]D22: +74° (c = 2.5, CHCl3); IR (neat, cm-1): 709, 1110, 1257, 1380, 1712; 1H NMR (200 MHz, CDCl3) δ = 1.43 (d, 3H, J = 6.2 Hz, C-6), 1.91 (s, 3H, CH3 Ac), 2.57 (s, 1H, OH-4), 3.42 (t, 1H, J = 9.2 Hz, H-4), 3.62 – 3.69 (m, 1H, H-5), 4.27 – 4.33 (m, 1H, H-2), 5.60 (t, 1H, J = 9.6 Hz, H-3), 5.70 (d, 1H J = 10.8 Hz, H-1), 7.24 – 7.74 (m, 9H, H Arom); 13C NMR (50 MHz, CDCl3) δ = 17.8 (C-6), 20.4 (CH3 Ac), 53.9, 74.2, 76.2, 82.5 (C-1, C-2, C-3, C-4, C-5), 123.3 – 133.9 (CH Arom), 130.8 (Cq SPh or NPhth), 131.3 (Cq SPh or NPhth), 131.6 (Cq SPh or NPhth), 167.0 (C=O NPhth), 167.7 (C=O NPhth), 171.1 (C=O Ac); HRMS: [M+Na]+ calcd for C22H21O6NSNa 450.09818, found 450.09792. Phenyl 3-O-acetyl-4-azido-2-phthalimido-2,4,6-trideoxy-1-thio-β-D-galactopyranoside (21): A solution of 27.4 g compound 20 (64 mmol) in 320 mL DCM and 25.8 mL pyridine (320 AcO SPh mmol, 5 equiv.) was cooled to -78˚C. Then 15.9 mL Tf2O (96 mmol, 1.5 equiv.) was added. NPhth The reaction mixture was kept at -40°C for 2h and allowed to warm to room temperature over a period of 12h. The reaction was quenched by addition of 50 mL MeOH. The mixture was diluted with 800 mL Et2O and N3

O

washed 4 times with 100 mL H2O, twice with 200 mL sat. CuSO4 and 200 mL brine. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Co-evaporation with toluene afforded the crude triflate intermediate as a colorless oil. The crude triflate was dissolved in 320 mL DMF, followed by the addition of 83.2 g NaN3 (1280 mmol, 20 equiv.) was added. The mixture was stirred at room temperature for 1.5h. The mixture was diluted with 800 mL EtOAc and washed three times with 200 mL H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 25.5 g compound 21 (56.3 mmol, 88%) as a white solid. TLC: 20% EtOAc/PE; [α]D22: -4° (c = 1, CHCl3); IR (neat, cm-1): 709, 864, 1049, 1126, 1272, 1380, 1581, 1705, 2113, 2931; 1H NMR (400 MHz, CDCl3) δ = 1.40 (d, 3H, J = 6.0 Hz, C-6), 1.95 (s, 3H, CH3 Ac), 3.91 – 3.96 (m, 1H, H-5), 3.97 (d, 1H, J = 3.2 Hz, H-4), 4.62 (t, 1H, J = 10.8 Hz, H-2), 5.60 (d, 1H, J = 10.4 Hz, H-1), 5.83 (t, 1H, J = 6.8 Hz, H3), 7.23 – 7.90 (m, 9H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 17.8 (C-6), 20.3 (CH3 Ac), 50.0 (C-2), 63.5 (C-4), 71.4 (C-3), 73.4 (C-5), 83.5 (C-1), 123.4 – 134.2 (CH Arom), 131.2 (Cq SPh), 131.5 (Cq NPhth), 131.8

131   

131

Chapter 8    (Cq NPhth), 167.0 (C=O NPhth), 168.0 (C=O NPhth), 169.94 (C=O Ac); HRMS: [M+NH4]+ calcd for C22H24O5N5S 470.14927, found 470.14810. Phenyl

N3

4-azido-2-phthalimido-3-O-tert-butyldimethylsilyl-2,4,6-trideoxy-1-thio-β-D-

O

galactopyranoside (22): To a solution of 25.5 g compound 21 (56.3 mmol) in 300 mL TBSO SPh NPhth MeOH was added Amberlite IR120 H+-resin. After 4 days stirring under reflux, the reaction mixture was filtered over Hyflo and concentrated under reduced pressure. The crude oil was dissolved in 250 mL DMF followed by the addition of 16.6 g TBDMSCl (110 mmol, 2.0 equiv.) and 7.48 g imidazole (110 mmol, 2.0 equiv.). The mixture was stirred at ambient temperature for 4 days. The reaction was quenched by the addition of 5 mL MeOH. The mixture was diluted with 500 mL EtOAc and washed with 250 mL 1M HCl and 250 mL sat. aq. NaHCO3. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Crystallization from boiling ethyl acetate afforded 19.3 g of compound 22 (40 mmol, 66%) as white, amorphous crystals. TLC: 20% EtOAc/PE; [α]D22: +14° (c = 1.8, CHCl3); IR (neat, cm-1): 709, 864, 1049, 1126, 1272, 1380, 1581, 1705, 2113, 2931; 1H NMR (200 MHz, CDCl3) δ = -0.25 (s, 3H, CH3 TBDMS), 0.00 (s, 3H, CH3 TBDMS), 0.71 (s, 9H, tBu TBDMS), 1.38 (d, 3H, J = 6.2 Hz, C-6), 3.64 (d, 1H, J = 2.8 Hz, H-3), 3.80 – 3.89 (m, 1H, H-5), 4.55 (t, 1H, J = 10.2 Hz, H-4), 4.71 (t, 1H, J = 10.2 Hz, H-2), 5.52 (d, 1H, J = 10.6 Hz, H-1), 7.20 – 7.75 (m, 9H, H Arom); 13C NMR (50 MHz, CDCl3) δ = -4.8 (CH3 TBDMS), 17.4 (C-6), 18.0 (Cq tBu TBDMS), 25.2 (tBu TBDMS), 52.6 (C-4), 66.7, 71.5, 73.3, 83.6 (C-1, C-2, C-3, C-5), 123.0 – 128.7 (CH Arom), 131.6 (Cq SPh), 132.0 (Cq NPhth), 132.6 (Cq NPhth), 167.3 (C=O, NPhth), 168.4 (C=O, NPhth); HRMS: [M+Na]+ calcd for C26H32O4N4SSiNa 547.18057, found 547.17974. CbzHN TBSO

O

SPh

Phenyl 4-(N-benzyloxycarbonyl)-amino-2-phthalimido-3-O-tert-butyldimethylsilyl-2,4,6trideoxy-1-thio-β-D-galactopyranoside (23): To a solution of 19.37 g compound 22 (40

mmol) in 120 mL toluene was added 80 mL of 1M Me3P solution in toluene (80 mmol, 2.0 equiv.) and 5 mL of H2O. The mixture was stirred overnight at ambient temperature. The mixture was concentrated under reduced pressure and traces of H2O where removed by co-evaporation with toluene. The crude oil was dissolved in 200 mL DCM and 14.9 g of N-(benzyloxycarbonyloxy)succinimide (60 mmol, 1.5 equiv.) and 6.63 mL of NEt3 (50 mmol, 1.25 equiv.) were added. The mixture was stirred for 5h at ambient NPhth

temperature. The reaction was quenched upon addition of 50 mL MeOH followed by concentration of the reaction mixture under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 22.8 g of compound 23 (35.2 mmol, 88%) as a white solid. TLC: 20% EtOAc/PE; [α]D22: +80° (c = 1, CHCl3); IR (neat, cm-1): 694, 864, 1018, 1103, 1242, 1380, 1527, 1689, 2090, 2947; 1H NMR (400 MHz, CDCl3) δ = -0.43 (s, 3H, CH3 TBDMS), 0.04 (s, 3H, CH3 TBDMS), 0.63 (s, 9H, tBu TBDMS), 1.28 (d, 3H, J = 6 Hz, C-6), 3.89 (td, 1H, J = 6.4 Hz, H-5), 4.03 (t, 1H, J = 5.6 Hz, H-4), 4.11 (t, 1H, J = 10.4 Hz, H-2), 4.64 (t, 1H, J = 5.2 Hz, H-3), 5.04 – 5.15 (m, 3H, CH2 and NH Cbz), 5.57 (d, 1H, J = 10.8 Hz, H-1), 7.22 – 7.86 (m, 9H H Arom); 13C NMR (100 MHz, CDCl3) δ = -5.6 (CH3 TBDMS), -4.8 (CH3 TBDMS), 17.3 (Cq tBu TBDMS), 19.1 (C-6), 25.2 (tBu TBDMS), 53.8 (C-4), 55.7 (C-2), 66.8 (CH2 Cbz), 68.8, 74.5, 83,4 (C-1, C-3, C-5), 123.0 – 134.3 (CH Arom), 131.5 (Cq NPhth), 131.7 (Cq NPhth), 132.4 (Cq SPh), 136.4 (Cq Cbz), 156.8 (C=O Cbz), 167.3 (C=O NPhth), 168.4 (C=O NPhth); HRMS: [M+H]+ calcd for C34H41O6N2SSi 633.24491, found 633.24481. 4-(N-benzyloxycarbonyl)-amino-2-azido-3-O-tert-butyldimethylsilyl-2,4,6-tri-deoxy-1-thio-β-Dgalactopyranoside (24): To a solution of 22.8 g compound 23 (35.2 mmol) in 800 mL n-

Phenyl CbzHN O

butanol was added 280 mL of ethylenediamine (4.2 mol, 120.0 equiv.). The mixture was heated overnight at 70˚C followed by concentration under reduced pressure. The crude oil was co-evaporated several times with toluene. The crude oil was dissolved in a solution of 260 mL MeOH and TBSO

N3

132   

132

SPh

Zwitterionic Polysaccharide PSA1    131 mL H2O followed by the addition of 7.2 g K2CO3 (72.7 mmol, 2 equiv.) and 55 mg CuSO4 (0.34 mmol, cat.). The reaction mixture was treated with a freshly prepared solution of triflyl azide in DCM [20.4 mL Tf2O (123 mmol) was added dropwise to a vigorously stirred biphasic mixture of 40 g NaN3 (615 mmol) in 154 mL H2O and 260 mL DCM at 0°C. The organic phase was separated and added to the reaction mixture mentioned above.] The mixture was stirred overnight at ambient temperature, diluted with 250 mL EtOAc and washed twice with 150 mL H2O The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash column chromatography using ethyl acetate/petroleum ether afforded 22.8 g of compound 24 (35.2 mmol, 88%) as a yellow oil. TLC: 20% EtOAc/PE; [α]D22: +1° (c = 1, CHCl3); IR (neat, cm-1): 694, 864, 1018, 1103, 1242, 1380, 1527, 1689, 2090, 2947; 1H NMR (400 MHz, CDCl3) δ = 0.12 (s, 3H, CH3 TBDMS), 0.15 (s, 3H, CH3 TBDMS), 0.87 (s, 9H, tBu TBDMS), 1.23 (d, 3H, J = 6.4 Hz, C-6), 3.07 (t, 1H, J = 9.6 Hz, H2), 3.58 – 3.63 (m, 2H, H-3, H-5), 3.89 (t, 1H, J = 6 Hz, H-4), 4.35 (d, 1H, J = 10.0 Hz, H-1), 4.73 (d, 1H, J = 10.4 Hz, NH Cbz), 5.07 (s, 2H, CH2 Cbz), 7.25 – 7.57 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = -5.6 (CH3 TBDMS), -4.8 (CH3 TBDMS), 16.7 (C-6), 17.9 (Cq tBu TBDMS), 25.6 (tBu TBDMS), 55.3 (C-4), 64.3 (C-2), 67.0 (CH2 Cbz), 74.0, 74.4, 86.6 (C-1, C-3, C-5), 127.6 – 133.1 (CH Arom), 131.9 (Cq SPh), 136.4 (Cq Cbz), 156.6 (C=O Cbz); HRMS: [M+H]+ calcd for C26H37O4N4SSi 529.22993, found 529.22874. CbzHN O

AcO

SPh

Phenyl 3-O-acetyl-4-(N-benzyloxycarbonyl)-amino-2-azido-2,4,6-trideoxy-1-thio-β-Dgalactopyranoside (7): To a mixture of 22.8 g of compound 24 (35 mmol) was added 175

mL MeOH and a catalytical amount of KOtBu. The mixture was stirred for 2h, neutralized by Amberlite IR120 H+-resin and filtered. The reaction mixture was concentrated under reduced pressure. The crude oil was dissolved in 130 mL pyridine and 45 mL Ac2O. The mixture was stirred for 5h and quenched with MeOH. The solvents were removed under reduced pressure followed by flash column chromatography using ethyl acetate/petroleum ether affording 14.3 g of compound 7 (31.5 mmol, 90%) as a N3

yellow oil. TLC: 20% EtOAc/PE; [α]D22: -22° (c = 1, CHCl3); IR (neat, cm-1): 1041, 1072, 1222, 1514, 1702, 1747, 2111; 1H NMR (400 MHz, CDCl3) δ = 1.25 (d, 3H, J = 6.0 Hz, H-6), 1.98 (s, 3H, CH3 Ac), 3.45 (t, 1H, J = 10.4 Hz, H-2), 3.73 (m, 1H, J = 10.0 Hz, J = 6.0 Hz, H-5), 4.18 (dd, 1H, J = 10.0 Hz, J = 3.6 Hz, H-4), 4.48 (d, 1H, J = 10.4 Hz, H-1), 4.83 (dd, 1H, J = 10.4 Hz, J = 3.6 Hz, H-3), 5.04 (d, 1H, J = 10.0 Hz, NH), 5.08 (d, 1H, J = 12.4 Hz, CHHPh), 5.16 (d, 1H, J = 12.4 Hz, CHHPh), 7.28 – 7.60 (m, 10H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 16.6 (C-6), 20.4 (CH3 Ac), 51.8 (C-4), 59.5 (C-2), 66.7 (CH2 Cbz), 73.5 (C-5), 74.4 (C-3), 86.4 (C1), 127.6 – 136.2 (CH Arom), 131.2 (Cq SPh), 136.2 (Cq Cbz), 156.3 (C=O Cbz), 169.8 (C=O Ac); HRMS: [M+H]+ calcd for C22H25O5N4S 457.15402, found 457.15353. 2-Azido-4,6-O-benzylidene-3-O-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-

Ph O O BzO

H

O

O

BzO

O N3

BzO

OBz

OH

2-deoxy-α/β-D-galactopyranoside (14): A solution of 117 mg donor 3 (0.2 mmol), 113 mg Ph2SO (0.56 mmol, 2.8 equiv.) and 150 mg TTBP (0.6 mmol, 3 equiv.) in 7 mL DCM was stirred over 100 mg activated MS3Å for 30min. The mixture was cooled to -60°C before 46 µL triflic acid anhydride (0.28 mmol, 1.4 equiv.) was added. The mixture was allowed to warm to -40°C in 1h followed by

addition of 59 mg acceptor 5 (0.2 mmol, 1 equiv.) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to room temperature over a period of 16h. The reaction mixture was diluted with 25 mL EtOAc and washed with 15 mL water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography using ethyl acetate/petroleum ether afforded 104 mg of the title compound 14 (0.12 mmol, 60%) as a colorless oil. TLC: 20% EtOAc/PE. IR (neat, cm-1): 702, 979, 1041, 1095, 1257, 1720, 2113, 2939; α-anomer: 1H NMR (600 MHz, CDCl3): 3.79 (s, 1H, H-5), 3.83 (dd, 1H, J = 12.6 Hz, J = 1.8 Hz, H-6), 3.98 (dd, 1H, J = 10.8 Hz, J = 3.6 Hz, H-2), 4.06 (dd, 1H, J = 12.6 Hz, J = 1.2 Hz, H-6), 4.18

133   

133

Chapter 8    (dd, 1H, J = 10.8 Hz, J = 2.4 Hz, H-3), 4.39 (d, 1H, J = 2.4 Hz, H-4), 4.60 – 4.63 (m, 2H, H-5’, H-6’), 4.67 (dd, 1H, J = 5.4 Hz, J = 3.0 Hz, H-4’), 5.32 (d, 1H, J = 3.6 Hz, H-1), 5.42 (s, 1H, CHPh), 5.56 (s, 1H, H-1’), 5.97 – 6.01 (m, 2H, H-2’, H-3’), 7.19 – 7.90 (m, 20H, H Arom); 13C NMR (100 MHz, CDCl3) δ = 59.2 (C-2), 62.5 (C5), 63.5 (C-6’), 69.1 (C-6), 70.1 (C-2’ and C-3’), 75.6 (C-4), 75.8 (C-3), 82.0, 82.3 (C-4’, C-5’), 92.4 (C-1), 100.3 (CHPh), 107.4 (C-1’), 125.7 – 133.4 (CH Arom), 128.7 (Cq Bz), 128.8 (Cq Bz), 129.1 (Cq Bz), 129.3 (Cq Bz), 137.3 (Cq benzylidene), 165.6 (2xC=O, Bz), 165.7 (C=O Bz), 166.2 (C=O Bz); HRMS: [M+Na]+ calcd for C47H41O14N3Na 894.24807, found 894.26489 Ph O BzO

H

O

O

BzO

O O N3

BzO

OBz O

O

OBz MeOOC

O

O

O

Me

Isopropyl 2-O-benzoyl-3-O-(2-azido-4,6-O-benzylidene-3-O-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-2deoxy-α-D-galactopyranosyl)-4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-β-D-galactopyranoside (15): One-pot approach: A solution of 89 mg donor 3 (0.15 mmol), 71 mg Ph2SO (0.35 mmol, 2.3 equiv.) and 74 mg TTBP (0.3 mmol, 2 equiv.) in 5 mL DCM was stirred over 100 mg activated MS3Å for 30min. The mixture was cooled to -60°C before 30 µL triflic acid anhydride (0.18 mmol, 1.2 equiv.) was added. The mixture was allowed to warm to -40°C in 1h followed by addition of 44 mg acceptor 5 (0.15 mmol, 1 equiv.) in 1 mL DCM/dioxane (10/1). Stirring was continued and the reaction mixture was allowed to warm to room temperature over a period of 16h. Subsequently the reaction was cooled back to -60°C followed by the addition of 35 µL triflic acid anhydride (0.2 mmol, 1.3 equiv.). The mixture was allowed to warm to -40°C in 1h followed by addition of 103 mg acceptor 6 (0.25 mmol, 1.7 equiv.) and 74 mg TTBP (0.3 mmol, 2 equiv.) in 1 mL DCM. Stirring was continued and the reaction mixture was allowed to warm to room temperature over a period of 16h. The reaction mixture was diluted with 25 mL EtOAc and washed with 15 mL water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography using ethyl acetate/petroleum ether afforded 118 mg of the title compound 15 (0.93 mmol, 62%) as a colorless oil. NIS/TMSOTf: A solution of 51 mg donor 1329 (0.05 mmol), 21 mg acceptor 6 (0.08 mmol, 1.5 equiv.) and 11 mg NIS (0.05 mmol, 1 equiv.) in 0.3 mL DCM was stirred over 10 mg MS4Å for 30min. The mixture was cooled to 0ºC before 2 µL TMSOTf (cat.) was added. The mixture was allowed to warm to ambient temperature over 1h. The reaction was quenched by adding 0.1 mL NEt3, diluted with 20 mL EtOAc and washed with 5 mL 1M Na2S2O4. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography using ethyl acetate/petroleum ether afforded 41 mg of the pure trisaccharide 15 (0.032 mmol, 65%) as a colorless oil. Sequential approach: Trisaccharide 15 was obtained from disaccharide 14 and acceptor 6 according to the procedure described for the conversion of donor 3 and acceptor 5 into disaccharide 14. TLC: 20% EtOAc/toluene; [α]D22: +80° (c = 1, CHCl3); IR (neat, cm-1): 702, 979, 1041, 1095, 1257, 1720, 2113, 2939; 1H NMR (600 MHz, CDCl3) δ = 1.05 (d, 3H, J = 6.0 Hz, CH3 isoprop), 1.21 (d, 3H, CH3 isoprop), 1.67 (s, 3H, CH3), 3.86 (d, 1H, J = 12.0 Hz, H-6), 3.39 (s, 1H, H-5’), 3.47 (s, 1H, H-5), 3.82 (s, 3H, CH3 COOMe), 3.83 – 3.85 (m, 2H, J = 12.0 Hz, J = 10.2 Hz, J = 3.0 Hz, H-2’, H-6), 3.92 – 3.98 (m, 2H, H-3, CH isoprop), 4.03 (d, 1H, J = 13.8 Hz, H-6’), 4.09 (d, 1H, J = 13.8 Hz, H-6’), 4.14 (d, 1H, J = 3.6 Hz, H-4’), 4.20 (dd, 1H, J = 10.8 Hz, J = 3.0 Hz, H-3’), 4.35 (d, 1H, J = 3.6 Hz, H-4), 4.57 – 4.62 (m, 3H, H-4’’, 2xH-6’’), 4.63 – 4.67 (m, 2H, H1, H-3’’), 5.18 (d, 1H, J = 3.6 Hz, H-1’), 5.29 (s, 1H, CHPh), 5.55 (t, 1H, J = 9.6 Hz, J = 8.4 Hz, H-2), 5.61 (d, 1H, J = 4.8 Hz, H-2’’), 5.66 (s, 1H, H-1’’), 6.00 – 6.02 (m, 1H, H-5’’), 6.92 – 8.08 (m, 30H, H Arom); 13C NMR

134   

134

Zwitterionic Polysaccharide PSA1    (150 MHz, CDCl3) δ = 21.8 (CH3 isoprop), 23.2 (CH3 isoprop), 25.5 (CH3), 52.6 (CH3 COOMe), 57.8 (C-2’), 62.8 (C-5), 63.4 (C-6’’), 65.2 (C-6’), 65.6 (C-5’), 67.6 (C-4), 68.9 (C-6), 69.9 (C-2), 72.2 (CH isoprop), 74.7 (C3’), 75.5 (C-4’), 75.8 (C-3), 77.2 (C-2’’), 81.8 (C-3’’), 81.9 (C-4’’), 96.6 (C-1’), 98.7 (Cq pyruvate), 99.6 (C-1), 100.1 (CHPh), 107.5 (C-1’’), 127.9 – 133.5 (CH Arom), 128.6 (Cq Bz), 128.7 (Cq Bz), 128.8 (Cq Bz), 129.0 (Cq Bz), 129.1 (Cq Bz), 137.2 (Cq benzylidene), 164.8 (C=O Bz), 164.9 (C=O Bz), 165.0 (C=O Bz), 165.5 (C=O Bz), 165.9 (C=O Bz), 170.3 (C=O COOMe); 13C-GATED NMR (150 MHz, CDCl3) δ = 96.6 (JC1’,H1’ = 172 Hz, C-1’), 99.6 (JC1,H1 = 156 Hz, C-1), 107.5 (JC1’’,H1’’ = 179 Hz, C-1’’); HRMS: [M+Na]+ calcd for C67H65O22N3Na 1286.39519, found 1286.38524. OH OH HO

H

O

O

HO

O AcHN

HO

OH O

O

OH HOOC

O

O

O

Me

Isopropyl 3-O-(2-acetamido-3-O-(β-D-galactofuranosyl)-2-deoxy-α-D-galactopyranosyl)-4,6-O-[1-(R)(carbonyl)-ethylidene]-β-D-galactopyranoside (16): Trisaccharide 15 (40 mg, 0.032 mmol) was dissolved in 0.5 mL of a 1M solution of Me3P in toluene (0.5 mmol, 15 equiv.) and 50 µL water. The solution was stirred for 16h and concentrated under reduced pressure. The residual oil was dissolved in 0.4 mL pyridine and 0.1 mL Ac2O and stirred for another 16h. The reaction mixture was diluted with 10 mL EtOAc and washed subsequently with 10 mL HCl (1M) and 10 mL sat. aq. NaHCO3. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. The residual oil was suspended in MeOH and treated with a catalytic amount of TsOH (~1mg). Stirring was allowed for 16h followed by the addition of 0.1 mL NEt3. The reaction mixture was concentrated under reduced pressure and filtered over a short plug of silica gel using ethyl acetate/light petroleum. The combinaed product-containing fractions were evaporated to dryness and dissolved in 0.2 mL H2O and 0.2 mL THF. This mixture was treated with 1.5 mL of a solution of 0.2M KOH in H2O until all ester groups were cleaved (checked by LCMS). The reaction mixture was acidified to pH = 5 using AcOH and concentrated under reduced pressure. Gel filtration (HW-40, 0.15M Et4NOAc in H2O) of the residual oil afforded the desired trisaccharide 16 (10 mg, 16 µmol, 50% over 4 steps) as a white foam. TLC: 20% EtOAc/toluene; 1H NMR (600 MHz, CDCl3) δ = 1.15 (d, 3H, J = 6.0 Hz, CH3 isoprop), 1.24 (d, 3H, J = 6.0 Hz, CH3 isoprop), 1.67 (s, 3H, CH3), 2.00 (s, 3H, CH3 NHAc), 3.51 (s, 1H, H-5), 3.58 (dd, 1H, J = 11.4 Hz, J = 4.0 Hz, H-6’’), 3.61 – 3.65 (m, 2H, J = 4.2 Hz, J = 4.0 Hz, H-2, H-6’’), 3.68 (m, 2H, H-6’), 3.72 (dd, 1H, J = 9.6 Hz, J = 4.0 Hz, H-3), 3.73 – 3.76 (m, 1H, H-5’’), 3.88 (d, 1H, J = 13.2 Hz, H-6), 3.95 (d, 1H, J = 13.2 Hz, H-6), 3.96 (s, 2H, H-3’’, H-4’’), 3.99 (s, 1H, H-2’’), 4.02 (dd, 1H, J = 10.8 Hz, J = 2.8 Hz, H-3’), 4.05 – 4.11 (m, 3H, H-4’, H-5’, CH isoprop), 4.35 (dd, 1H, J = 11.4 Hz, J = 2.4 Hz, H-2’), 4.38 (d, 1H, J = 3.6 Hz, H-4), 4.51 (d, 1H, J = 7.8 Hz, H-1), 5.03 (d, 1H, J = 1.8 Hz, H-1’’), 5.23 (d, 1H, J = 3.6 Hz, H-1’); 13C NMR (150 MHz, CDCl3) δ = 21.8 (CH3 isoprop), 23.2 (2xCH3 isoprop and NHAc), 26.0 (CH3 pyruvate), 49.1 (C-2’), 61.9 (C-6’), 63.6 (C6’’), 65.9 (C-6), 66.9 (C-5), 68.3 (C-4), 69.6 (C-5’), 69.8 (C-2), 71.7 (C-5’’), 71.8 (C-4’), 73.8 (CH isoprop), 76.1 (C-3), 76.7 (C-3’), 77.9 (C-4’’), 82.4 (C-2’’), 83.7 (C-3’’), 94.9 (C-1’), 95.1 (Cq pyruvate), 101.2 (C-1), 110.1 (C-1’’); 13C-GATED NMR (150 MHz, CDCl3) δ = 94.9 (JC1’,H1’ = 174 Hz, C-1’), 101.2 (JC1,H1 = 156 Hz, C-1), 110.1 (JC1’’,H1’’ = 176 Hz, C-1’’). ESI-MS: 680.2 [M+Na]+.

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135

Chapter 8    HO BzO

H

O

OBn O

O

BzO

N3

OBz O

O

OBz

BzO

O

O O

MeOOC

Me

Isopropyl 2-O-benzoyl-3-O-(2-azido-6-O-benzyl-3-O-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-2-deoxyα-D-galactopyranosyl)-4,6-O-[1-(R)-(methoxycarbonyl)-ethylidene]-β-D-galactopyranoside (26): Compound 15 (100 mg, 0.08 mmol) and 68 µL Et3SiH (0.43 mmol, 5 equiv.) were dissolved in 2 mL DCM and stirred over 50 mg activated MS3Ǻ. The mixture was cooled to -78°C and 30 µL triflic acid (0.34 mmol, 4 equiv.) was added. The reaction mixture was allowed to warm to -50°C until TLC indicated complete conversion (30min). The reaction was subsequently quenched by adding 0.4 mL MeOH and 0.3 mL NEt3 (in this order!), diluted with 15 mL EtOAc and washed with H2O. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Flash chromatography using ethyl acetate/petroleum ether afforded 93 mg of trisaccharide 26 (0.074 mmol, 92%) as a colorless oil. TLC: 40% EtOAc/PE; [α]D22: +44° (c = 1, CHCl3); IR (neat, cm-1): 709, 1049, 1103, 1265, 1450, 1720, 2113, 2877; 1H NMR (600 MHz, CDCl3) δ = 1.04 (d, 1H, J = 6.6 Hz, CH3 isoprop), 1.20 (d, 1H, J = 6.6 Hz, CH3 isoprop), 1.64 (s, 3H, CH3), 2.74 (bs, 1H, OH-4’), 3.33 (d, 1H, J = 0.6 Hz, H-5), 3.37 (dd, 1H, J = 10.8 Hz, J = 4.0 Hz, H-6’), 3.48 (dd, 1H, J = 10.2 Hz, J = 6.6 Hz, H-6’), 3.70 (dd, 1H, J = 10.2 Hz, J = 3.6 Hz, H-2’), 3.74 (bs, 1H, H-4’), 3.79 (s, 3H, CH3 COOMe), 3.82 – 3.86 (m, 1H, J = 7.2 Hz, J = 6.0 Hz, H-5’), 3.92 – 4.00 (m, 4H, H-3, H-6, H-3’, CH isoprop), 4.12 (dd, 1H, J = 13.2 Hz, J = 1.2 Hz, H-6), 4.36 (d, 1H, J = 4.2 Hz, H-4), 4.40 (d, 1H, J = 12.0 Hz, CHHPh), 4.45 (d, 1H, J = 12.0 Hz, CHHPh), 4.52 (d, 1H, J = 12.0 Hz, J = 6.6 Hz, H-6’’), 4.57 (d, 1H, J = 8.4 Hz, H-1), 4.60 – 4.64 (m, 1H, J = 12.0 Hz, J = 4.2 Hz, H-4’’, H-6’’), 5.29 (d, 1H, J = 3.0 Hz, H-1’), 5.55 (dd, 1H, J = 12.6 Hz, J = 8.4 Hz, H-2), 5.58 (s, 1H, H-1’’), 5.61 – 5.65 (m, 2H, H-2’’, H-3’’), 5.94 – 5.97 (m, 1H, H-5’’),7.88 – 8.06 (m, 30H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 19.4 (CH3 isoprop), 21.8 (CH3 isoprop), 23.2 (CH3), 52.6 (CH3 COOMe), 57.9 (C-2’), 62.9 (C-6’’), 65.4 (C-6), 65.6 (C-5), 66.5 (C-4), 68.9 (C-5’), 69.5 (C-4’),69.9 (C-6’), 70.2 (C-5’’), 70.3 (C-2), 72.3 (CH isoprop), 73.3 (CH2 Bn), 73.4 (C-3), 75.8 (C-3’), 77.2 (C-2’’ or C-3’’), 81.7 (C-3’’ or C-2’’), 82.0 (C-4’’), 83.9 (C-1’), 98.8 (Cq pyruvate), 99.7 (C-1), 107.2 (C-1’’), 125.6 – 133.5 (CH Arom), 129.4 (Cq Bz), 129.6 (Cq Bz), 129.7 (Cq Bz), 129.9 (Cq Bz), 137.9 (Cq Bn), 164.8 (C=O Bz), 165.1 (C=O Bz), 165.5 (C=O Bz), 165.9 (C=O Bz), 170.4 (C=O COOMe); 13C-GATED NMR (150 MHz, CDCl3) δ = 83.9 (JC1’,H1’ = 171 Hz, C-1’), 99.7 (JC1,H1 = 158 Hz, C-1), 107.2 (JC1’’,H1’’ = 175 Hz, C-1’’); HRMS: [M+H]+ calcd for C67H69O22N3 1266.42890, found 1266.43420. TCAHN O AcO N3 BzO

H

O

O

O

BzO

OBn O N3

BzO

OBz O

O

OBz MeOOC

O

O

O

Me

Isopropyl 2-O-benzoyl-3-O-(2-azido-6-O-benzyl-3-O-(2,3,5,6-tetra-O-benzoyl-β-D-galactofuranosyl)-4-O-(3-Oacetyl-2-azido-4-trichloroacetamido-2,4,6-trideoxy-β-D-galactopyranosyl)-2-deoxy-α-D-galactopyranosyl)-4,6O-[1-(R)-(methoxycarbonyl)-ethylidene]-β-D-galactopyranoside (2): A solution of 21 mg donor 8 (0.055 mmol), 28 mg Ph2SO (0.14 mmol, 2.5 equiv.) and 17 mg tri-tert-butylpyrimidine (0.07 mmol, 1.3 equiv.) in 2.5 mL DCM was stirred over 50 mg activated MS3Å for 30min. The mixture was cooled to -60°C before 12 µL triflic 136   

136

Zwitterionic Polysaccharide PSA1    acid anhydride (0.07 mmol, 1.3 equiv.) was added. The mixture was allowed to warm to -40°C over 1h followed by addition of 60 mg trisaccharide acceptor 26 (0.047 mmol, 0.85 equiv.) Stirring was continued and the reaction mixture was allowed to warm to 0°C. The reaction mixture was diluted with 15 mL EtOAc and washed with water. The organic phase was dried (MgSO4), filtered and concentrated under reduced pressure. Size exclusion chromatography (LH20, eluent DCM/MeOH 1/1) followed by preparative HPLC (column: Gemini C18 5µm, 21x150mm, eluent: A: 5% ACN/H2O + 0.1% TFA, B:100% ACN, 25 mL/min, gradient: 82%-92% (10min)) afforded 5 mg of the title compound 2 (3 µmol, 17%; 53% recovered trisaccharide 26) as a colorless oil. TLC: 40% EtOAc/PE; IR (neat, cm-1): 702, 979, 1041, 1095, 1257, 1720, 2113, 2939; 1H NMR (600 MHz, CDCl3) δ = 1.04 (d, 1H, J = 6.6 Hz, CH3 isoprop), 1.20 (d, 1H, J = 6.6 Hz, CH3 isoprop), 1.24 (d, 1H, J = 6.4 Hz, H-6), 1.61 (s, 3H, CH3), 2.05 (s, 3H, CH3 Ac), 3.13 (dd, 1H, J = 10.8 Hz, J = 3.6 Hz, H-2’’), 3.32 (bs, 1H, H-5), 3.51 – 3.53 (m, 1H, H-6’), 3.65 (dd, 1H, J = 11.4 Hz, J = 3.3 Hz, H-2’), 3.69 (dd, 1H, J = 9.0 Hz, J = 7.8 Hz, H-6’), 3.78 (s, 3H, CH3 COOMe), 3.90 – 3.94 (m, 1H, J = 6.0 Hz, CH isoprop), 3.95 – 3.97 (m, 3H, H-6, H-4’, H-5’), 4.01 (dd, 1H, J = 10.2 Hz, J = 3.6 Hz, H-3), 4.09 (dd, 1H, J = 10.8 Hz, J = 2.4 Hz, H-3’), 4.11 (d, 1H, J = 12.6 Hz, H-6), 4.33 (d, 1H, J = 11.4 Hz, CHHPh), 4.35 (d, 1H, J = 4.2 Hz, H-4), 4.35 (dd, 1H, J = 11.4 Hz, J = 4.0 Hz, H-4’’), 4.45 (d, 1H, J = 11.4 Hz, CHHPh), 4.48 (dd, 1H, J = 11.4 Hz, J = 7.6 Hz, H-6 fur.), 4.58 (d, 1H, J = 8.4 Hz, H1), 4.63 (q, 1H, J = 6.6 Hz, H-5’’), 4.67 (dd, 1H, J = 12.0 Hz, J = 4.6 Hz, H-6 fur.), 4.71 (m, 1H, J = 3.0 Hz, H-4 fur.), 4.76 (d, 1H, J = 3.6 Hz, H-1’’), 5.26 (dd, 1H, J = 11.4 Hz, J = 3.9 Hz, H-3’’), 5.35 (d, 1H, J = 3.0 Hz, H1’), 5.54 (dd, 1H, J = 9.6 Hz, J = 8.4 Hz, H-2), 5.61 – 5.63 (m, 2H, H-2 fur. and H-3 fur.), 5.69 (bs, 1H, H-1 fur.), 5.94 (m, 1H, J = 3.0 Hz, H-5 fur.), 6.59 (d, 1H, J = 9.2 Hz, NH-4’’), 7.17 – 8.12 (m, 30H, H Arom); 13C NMR (150 MHz, CDCl3) δ = 19.4 (CH3 isoprop), 21.8 (CH3 isoprop), 23.2 (CH3), 52.7 (CH3 COOMe), 53.1 (C4’’), 58.3 (C-2’’), 58.7 (C-2’), 63.2 (C-6’’), 64.5 (C-5’’), 65.4 (C-6), 65.6 (C-5), 66.4 (C-4), 67.5 (C-6’), 69.6 (C3’’), 69.7 (C-4’or C-5’), 70.5 (C-2 and C-5 fur.), 72.4 (CH isoprop), 73.2 (CH2 Bn), 73.2 (C-3), 74.5 (C-3’), 76.8 (C-5’or C-4’), 77.5 (C-2 fur. or C-3 fur.), 80.9 (C-4 fur.), 81.0 (C-3 fur. or C-2 fur.), 92.3 (Cq CCl3), 93.4 (C-1’), 98.2 (C-1’’), 98.9 (Cq pyruvate), 99.7 (C-1), 107.1 (C-1 fur.), 127.7 – 135.8 (CH Arom), 128.7 (Cq Bz), 128.9 (Cq Bz), 129.0 (Cq Bz), 129.5 (Cq Bz), 129.9 (Cq Bz), 137.8 (Cq Bn), 162.5 (C=O TCA), 164.9 (C=O Bz), 165.3 (C=O Bz), 165.6 (C=O Bz), 165.7 (C=O Bz), 166.0 (C=O Bz), 170.0 (C=O Ac or COOMe), 170.5 (C=O COOMe or Ac); HRMS: [M+Na]+ calcd for C77H78Cl3N7O26Na 1644.39543 found 1644.39543. X-ray crystallographic data Purified pyruvate 11 (200 mg) was suspended in 2 ml of absolute EtOH. The mixture was heated to 70°C within a few minutes and kept untill almost all solids had dissolved. The sealed vial then was cooled up to 15°C (speed: approximately 4°C per hour). After reaching 15°C, the mixture was warmed to 50°C, kept for several minutes at this temperature and again cooled to 15°C (4 degrees per hour). This process was repeated several times till crystals of suitable size were obtained. The needle like transparent crystal of approximately 0.3 x 0.15 x 0.08 mm size was measured on the Kappa CCD using MoKα radiation. The reflections were collected up to 2θ = 25°. The cell was obtained and refined from 25 frames from reflection within the same angle range using Scalepack30 software. The data was processed using Denzo package.30 The structure was solved using direct methods (SHELXS9-7).31 The full matrix least-squares refined was applied using SHELXL97 program.32 All of non-H atoms were found on Fourier difference maps and refined anisotropically while all H atoms were included from geometry and not refined. Table 1 lists crystallographic data for compound 11.

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Chapter 8    Table 1. Crystal data and structure refinement for pyruvate acetal 11. Empirical formula C30H30O8S Formula weight 550.60 Temperature 293(2) Wavelength 0.71073 Crystal system Orthorhombic Space group P 21 2 1 2 1 Unit cell dimensions 6.2350(4) 9.7600(7) 46.750(4) Volume 2844.9(4) Z 4 Density (calculated) 1.286 Absorption coefficient 0.162 F(000) 1160 Crystal size 0.3 x 0.15 x 0.06 Theta range for data collection 2.5 → 25 Reflections collected 7621 Independent reflections 4549 [Rint = 0.0755] Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4549 / 0 / 363 1.038 Goodness-of-fit on F2 Final R indices [I>2sigma(I)] R1 = 0.0706, wR2 = 0.1485 R indices (all data) R1 = 0.1300, wR2 = 0.1780 Absolute structure parameter -0.12(17) Extinction coefficient 0.011(3) Largest diff. peak and hole 0.258 and -0.184 e.Å-3

References and Notes 1

Published in part: Van den Bos, L.J.; Boltje, T.J.; Provoost, T.; Mazurek, J.; Overkleeft, H.S.; Van der Marel, G.A. submitted.

2

(a) Baumann, H.; Tzianabos, A.O.; Brisson, J.-R.; Kasper, D.L.; Jennings, H.J. Biochemistry 1992, 31, 4081–4089 (b) Tzianabos, A.O.; Pantosti, A.; Baumann, H.; Brisson, J.-R.; Jennings, H.J.; Kasper, D.L. J. Biol. Chem. 1992, 267, 18230–18235 (c) Tzianabos, A.O.; Finberg, R.W.; Wang, Y.; Chan, M.; Onderdonk, A.B.; Jennings, H.J.; Kasper, D.L. J. Biol. Chem. 2000, 275, 6733–6740 (d) Tzianabos, A.O.; Wang, J.Y.; Kasper, D.L. Carbohydr. Res. 2003, 338, 2531–2538.

3

Recent studies claimed that zwitterionic polysaccharides prevent abscess formation: (a) Tzianabos, A.O.; Finberg, R.W.; Wang, Y.; Chan, M.; Onderdonk, A.B.; Jennings, H.J.; Kasper, D.L. J. Biol. Chem. 2000, 275, 6733–6740. (a) Harding, C.V.; Roof, R.W.; Allen, P.M.; Unanue, E.R. Proc. Natl. Ac. Sci. USA 1991, 88, 2740–2744 (b) Abbas, A.K.; Lichtman, A.H.; Pober, J.S. in Cellular and Molecular Immunology, New York, W.B.

4

6

Saunders Company, 2000. (a) Tzianabos, A.O.; Kasper, D.L. Curr. Opin. Microbiol. 2002, 5, 92–96 (b) Kalka-Moll, W.; Tzianabos, A.O.: Bryant, P.W.; Niemeyer, M.; Ploegh, H.L.; Kasper, D.L. J. Immunol. 2002, 169, 6149–6153 (c) Cobb, B.A.; Wang, Q.; Tzianabos, A.O.; Kasper, D.L. Cell 2004, 117, 677–687. Kalka-Moll, W.; Tzianabos, A.O.: Wang, Y.; Carey, V.J.; Finberg, R.W.; Onderdonk, A.B.; Kasper, D.L. J.

7

Immunol. 2000, 164, 719–724. Tzianabos, A.O.; Onderdonk, A.B.; Rosner, B.; Cisneros, R.L.; Kasper, D.L. Science 1993, 262, 419–419.

5

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(a) Codée, J.D.C.; Litjens, R.E.J.N.; Van den Bos, L.J.; Overkleeft, H.S.; Van der Marel, G.A. Chem. Soc. Rev. 2005, 34, 769–782 (b) Demchenko, A.V. Lett. Org. Chem. 2005, 2, 580–589.

9

Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950. 10 Misra, A.K.; Agnihotri, G. Carbohydr. Res. 2004, 339, 885–890. 11 Chen, C.-T.; Weng, S.-S.; Kao, J.-Q.; Lin, C.-C.; Jan, M.-D. Org. Lett. 2005, 7, 3343–3346. 12 Nguyen, H.M.; Poole, J.L.; Gin, D.Y. Angew. Chem. Int. Ed. 2001, 40, 414–417. 13 Grundler, G.; Schmidt, R.R. Liebigs Ann. Chem. 1984, 1826–1847. 14 Malet, C.; Hindsgaul, O. Carbohydr. Res. 1997, 303, 51–66. 15 (a) Ziegler, T.; Eckhardt, E.; Herold, G. Tetrahedron Lett. 1992, 33, 4413–4416 (b) Ziegler, T. Tetrahedron Lett. 1994, 35, 6857–6860. 16 Crich, D., Smith, M., Yao, Q., Picione, J. Synthesis 2001, 323–326. 17 Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279. 18 Codée, J.D.C.; Litjens, R.E.J.N.; Den Heeten, R.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1519–1522. 19 Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020. 20 Van den Bos, L.J.; Dinkelaar, J.; Overkleeft, H.S.; Van der Marel, G.A. J. Am. Chem. Soc. 2006, 128, 13066–13067. 21 Veeneman, G.H.; Van Leeuwen, S.H.; Van Boom, J.H. Tetrahedron Lett. 1990, 31, 1331–1334. 22 Stereochemistry around the anomeric centers was assigned on the basis of 13C-GATED measurements: Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2 1974, 293–299. 23 Although dioxane is known as a α-directing solvent, β-linked disaccharide 19 was the only product found: (a) Demchenko, A.V.; Stauch, T.; Boons, G.-J. Synlett 1997, 818–820 (b) Demchenko, A.V.; Rousson, E.; Boons, G.-J. Tetrahedron Lett. 1999, 40, 6532–6526. 24 Tiwari, P.; Kumar Misra, A. Carbohydr. Res. 2006, 341, 339–350. 25 Medgyes, A.; Farkas, E.; Lipták, A.; Pozsgay, V. Tetrahedron 1997, 53, 4159–4178. 26 27 28 29

Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41, 5547–5551. See Chapter 7 for route of synthesis. Malet, C.; Hindsgaul, O. Carbohydr. Res. 1997, 303, 51–66. Codée, J.D.C.; Van den Bos, L.J.; Litjens, R.E.J.N.; Overkleeft, H.S.; Van Boom, J.H.; Van der Marel, G.A. Org. Lett. 2003, 5, 1947–1950.

30 Otwinowski, Z.; Minor, W. Methods in Enzymology Carter, C.W.; Sweet, R.M. eds, New York, Academic Press, 1997, 276, 307–326. 31 Sheldrick, G.M. SHELXS-97. Program for Crystal Structure Solution. University of G¨ottingen, Germany, 1997. 32 Sheldrick, G.M. SHELXL97. Program for the Refinement of Crystal Structures. University of G¨ottingen, Germany, 1997.

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Chapter 9 │  Summary,    Mechanistic Aspects, and      Perspective 

           

   

General Conclusion and Future Prospects This Thesis reports on research aimed at the assembly of acidic and zwitterionic polysaccharides of bacterial origin, using suitably protected 1-thioglycoside residues. Thioglycosides are attractive monosaccharide building blocks because of their high stability towards the diverse reaction conditions used in functional group manipulations. Recently, the use of 1-thioglycosides has been stimulated by the advent of the 1-benzenesulfinylpiperidine (BSP)/triflic anhydride (Tf2O) and the diphenylsulfoxide (Ph2SO)/Tf2O activator system. These sulfonium ion based activators proved to be efficient promoters for glycosylations in which inreactive (disarmed) 1-thioglycosides are employed. Furthermore, one-pot orthogonal and chemoselective oligosaccharide synthesis strategies that involve the use of 1thioglycosides in combination with the above mentioned sulfonium based activator systems have proven to be feasible. Chapter 1 presents an overview of the different procedures for the construction of acidic oligosaccharides. Broadly speaking two approaches can be

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Chapter 9   

distinguished, the first one involves introduction of the carboxylic acid function at the monosaccharide stage and the second introduction of the carboxylate at the oligosaccharide stage. Both approaches require an efficient oxidation method. Reports on the oxidation at the oligosaccharide level exceed in number those concerning the use of suitably protected 1thioglycuronic acid ester donors. The latter approach is an important component of the research described in this thesis. In Chapter 2 an efficient strategy for the synthesis of 1thioglycuronides is described. Using the TEMPO/BAIB reagent combination, a series of functionalized 4,6-unprotected 1-thioglycosides was in a chemo- and regioselective fashion converted into the corresponding 1-thioglycuronides. Despite the deactivating carboxylate ester present on the carbohydrate core, these 1-thioglycuronides could smoothly be activated using the Ph2SO/Tf2O or BSP/Tf2O reagent combination, as is exemplified in the chemoselective synthesis of a protected form of the trisaccharide portion present on the capsules of the fungus Fusarium sp. M7-1. Chapter 3 discusses the synthesis and application of 6,3-lactone derivatives of 1-thioglycuronides. These compounds were obtained from the corresponding 3,6-unprotected 1-thioglycosides via a tandem oxidation/lactonization process using the TEMPO/BAIB system. Furthermore, the synthesis of the [β-D-Galp-(1→3)-α-DGalpA-(1→4)-α-D-GlcN3-OMe] trisaccharide was achieved. The used strategy required a minimum number of protective group manipulations at the disaccharide stage. The central galacturonic acid was initially used as donor building block followed by base-mediated hydrolysis of the lactone at the disaccharide stage. In the next stage this acceptor disaccharide was coupled to the non-reducing end of a galactose building block. Chapter 4 describes the use of 1-thio functionalized mannuronic acid esters in oligosaccharide synthesis. Other then the corresponding 1-thioglucuronic and galacturonic acid esters, the mannuronic acid derivatives showed high to exclusive β-selectivity in the Ph2SO/Tf2O-mediated coupling procedure. The developed strategy was used in the first synthesis of an alginate trisaccharide. Chapter 5 describes the glycosylation properties of deactivated 1-thiomannosazide and 1-thio mannosaziduronic acid esters. Installation of an acetyl protective group at positions C4 and C6 of the mannosazide led to reasonable β-selectivities. Acetyl protection at C3 of these mannosazide donors yielded, irrespective of the protective group pattern of the donor glycoside, α-coupled products. Chapter 6 describes a tethered nucleophilic substitution approach for the synthesis of 4-amino functionalized galacto- and talopyranosides. Regioselective installation of a trichloroacetimidate at C6 of 4,6-unprotected gluco- and mannopyranosides followed by conversion of C4-OH into the triflate and base treatment delivered the corresponding 4-amine substituted galacto- and talopyranoside, respectively. Chapter 7 describes a convergent route of synthesis to a protected trisaccharide corresponding to the repeating unit of the zwitterionic polysaccharide Sp1 isolated from the cell-wall of Streptococcus pneumoniae. This type of zwitterionic polysaccharides was recently discovered as the main pathogen in the formation of intraabdominal abscesses. Global deprotection of the protected trisaccharide resulted in complex reaction mixtures. In

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Summary, Mechanistic Aspects, and Perspective    Figure 1. TCAHN

TCAHN D

AcO

O N3

BzO

O

I H

BzO

O

OH

A

BzO

1

B

HO

Me

OBn

2

O

MeO2C

N3

O

OH HO

O

II

OB z

N3 BzO

H

O

BzO O

C

3

O

D

AcO one-pot deh ydr ative glycosylatio n

O

A

Oi Pr

O

OBn O

B

N3

BzO

OBz O

C

O

OBz

OBz

4

MeOO C

O

OiP r

O

Me

particular, unmasking the N-trichloroacetyl group did not proceed uneventfully. Changing this protective group for an N-benzyloxycarbonyl group may circumvent these problems. Chapter 8 describes a synthetic study towards tetrasaccharide 4 corresponding to the repeating unit of the zwitterionic polysaccharide PSA1 isolated from the cell-wall of Bacteriodes fragilis. The ABC trisaccharide part was synthesized using an iterative one-pot dehydrative synthesis approach using the Ph2SO/Tf2O activator system (Figure 1). Unfortunately, the final condensation towards tetrasaccharide 4 proceeded in low yield most probably as a result of the steric environment around the acceptor nucleophile. Reversing the order of glycosylation events, first assembling disaccharide 2, which is then employed in the one-pot dehydrative glycosylation approach towards tetrasaccharide 4 may prove to be a more efficient strategy.

Mechanistic Aspects and Perspective

The research described in this Thesis shows that 1-thioglycuronides and sulfonium triflate activators are a promising combination for the assembly of complex oligosaccharides. As with other glycosylation procedures, the outcome of these glycosylation reactions in terms of yield and stereoselectivity1 can not always be predicted. Figure 2 depicts the postulated Figure 2. Postulated Mechanism for BSP/Tf2O and Ph2SO/Tf2O-Mediated Activation of 1-Thioglycosides 5.

COOP O (PO)n

R S OTf Ph OTf Ph

-60°C

α-triflate

contact ion pair

COOP O

COOP O

(PO)n

(PO)n

S

6

7

OTf R

R = piperidine (BSP) S OTf R = phenyl (Ph SO) 2 Ph

8 ROH -TfOH

COOP O

COOP O

(PO)n SPh 5

OTf

OTf

(PO)n OR 9

143   

143

Chapter 9   

mechanism for the pre-activation of 1-thioglycoside 5 using either the BSP/Tf2O or Ph2SO/Tf2O reagent combination. Initial attack of the anomeric sulfur group on in situ generated sulfonium bistriflate results in the formation of intermediate 6, which is believed to be rapidly converted into an equilibrium mixture of covalently bound α-triflate 7 and contact ion pair 8. Studies by the Crich laboratory indicate that the stereochemical outcome of these sulfonium ion-mediated glycosylations is governed by the position of this equilibrium. Table 1.

entry

donor

AcO

1

activator yield (α/β)b,c

acceptor

disaccharide AcO

OH

COO Me O

SPh

BnO

O

BnO BnO

BnO

OBn

11

10

O Me

Ph2SO/Tf2O 80% (1/2)

BnO

BnO

O

O BnO BnO 12

BnO OMe

O

O

2

CO OMe O

S Ph

O

RO OMe 11: R = Bn 14: R = Ac

OBn

13

O

RO RO

O

B nO

OH

Ph2SO/Tf2O 15: 69% (1/0) 16: 98% (1/0)

O

O O

BnO

O RO

OR

O Me

RO OBn 15: R = Bn 16: R = Ac

O SPh

O

3

BnO

O

Ph2SO/Tf2O 74% (1/0)

14

O Bn a

18

17 , α/ β = 1/1 13

O pMP

HO

4

13

COOMe O

B nO

O

O pMP

OB n 19

Ph2SO/Tf2O 50% (4/1)

O MeOO C O

O O

B nO

OBn

BnO OBn 20

O

5

13

HO BnO

CO OMe O BnO O Me 21

Ph2SO/Tf2O 91% (1/0)

O BnO

O

O BnO OBn

CO OMe O BnO O Me

22 a

Ph2SO, Tf2O, TTBP, -60°C then PhSH, rt, 58%. NMR spectroscopy.

144   

144

b

isolated yields.

c

anomeric ratios were determined by 1H

Summary, Mechanistic Aspects, and Perspective   

Electronic destabilization of the positive charge of the contact ion pair would shift the equilibrium to the α-triflate 7, thereby facilitating an SN2 type reaction with the incoming nucleophilic acceptor leading to higher β-selectivities in product 9.2,3,4 Another decisive factor on the anomeric selectivity is the reactivity of the acceptor nucleophile, which was implied previously by Paulsen.5 In his seminal review, it was stated that primary alcohol acceptors are considerably more reactive than their secondary counterparts and therefore react with less anomeric selectivity. Chapter 3 describes, amongst others, the Ph2SO/Tf2O-mediated glycosylation of phenyl 1thio-β-D-galacturonic acid lactones with a variety of acceptor molecules. Compared to the phenyl 1-thio-β-D-galacturonic acid esters described in Chapter 2, increased α-selectivities were observed (Table 1). Coupling methyl uronate donor 10 with acceptor 11 yielded an anomeric mixture of α/β = 1/2, whereas coupling of lactone donor 13 with the same acceptor proceeded with complete α-selectivity (entries 1 and 2). The altered steric and electronic environment has a beneficial effect on the α-selectivity. In a further experiment, it was revealed that the orientation of the thiophenyl functionality in lactone donor 17 does not influence the anomeric product ratio (entry 3). In order to identify the exact reactive intermediate in these galactosylations, pre-activation of the anomerically mixed donor 17 was monitored using low temperature NMR spectroscopy (Figure 3). Figure 3A depicts a detailed part of the spectrum of a mixture of donor 17 and Ph2SO at -55°C in CD2Cl2. Addition of Tf2O to the cooled NMR tube then showed conversion of the anomeric donor mixture into a single species (Figure 3B). Based on the altered value of the chemical shift of the anomeric signal (δ = 6.01 ppm) and the peak splitting pattern, β-triflate 23 is postulated as the actual glycosylating species. Upon addition of an acceptor nucleophile, β-triflate 23 is selectively displaced in an SN2-type manner leading to a α-coupled disaccharide. The predominant Figure 3. Low temperature NMR spectrum of donor 17 and Ph2SO before (A) and after (B) addition of Tf2O. CDHCl2

O

A

-55°C in CD2Cl2

SPh

O

CH2 (Bn)

O

BnO

1β 5α 5β

OBn

1α 3β

17 (α/β = 1/1) 6.4

6.2

6.0

5.8

5.6

5.4

5.2

5.0

4α

3α

4.8

4β 2β 2α

4.6

4.4

4.2

4.0

3.8

3.6

ppm

4.0

3.8

3.6

ppm

CDHCl2

O

B

O

BnO

-55°C in CD2Cl2

δ(C1) = 104.5 ppm 1 JC,H = 189 Hz

OTf

O

CH2 (Bn)

1

5

OBn

4 2

3

23 6.4

6.2

6.0

5.8

5.6

5.4

5.2

5.0

4.8

4.6

4.4

4.2

145   

145

Chapter 9   

presence of β-triflate 23 at the expense of the contact ion pair intermediate is most probably the result of the anomeric effect and of torsional strain preventing hybridization changes (favored sp3 → unfavored sp2) necessary for oxocarbenium ion formation.6 Formation of the α-triflate intermediate is unlikely due to the destabilizing Δ2-effect.7,8 Despite the presented high α-selectivities, the difference in the anomeric selectivity for the Ph2SO/Tf2O-mediated glycosylation of donor lactone 13 with galacturonic acceptor 19 (Table 1, entry 4) and the corresponding α-selective glucuronic acid acceptor 21 (entry 5) is still difficult to rationalize. It could well be that double stereodifferentation, first described by Spijker and Van Boeckel in the field of carbohydrate chemistry,9 is at the basis of the moderate anomeric selectivity obtained for galacturonic acceptor 19. Using L-galacturonic Figure 4. acid ester 24 having exactly the same O electronic effects but different OpMP O stereochemical preferences, when Op MP O MeO OC O HO OB n compared to 19, may support this theory OBn HO OBn (Figure 4). An alternative option may be D24 25 galacturonic acid lactone 25. In Chapters 4 and 5 the high β-selectivity of deactivated 1-thiomannuronic acid esters, 1thiomannosazides and 1-thiomannosaziduronic acid ester donors is described. It was reasoned that the strongly electron withdrawing carboxylate function dictates the equilibrium between the α-triflate and the contact ion pair to be completely shifted towards the α-triflate, favoring β-side attack. To investigate this, 1-thiomannuronic acid ester 26 was chosen as a model compound for variable temperature NMR experiments (Figure 5). Recording the 1H NMR spectra at +25°C and -50°C revealed the existence of a conformational mixture. On the basis Figure 5. 1H NMR spectrum of donor 26 at +25°C (A) and at -50°C (B). MeOO C

Me OOC OBn O AcO BnO S Ph

CDHCl2

A

33% 4 26a ( C 1)

+25°C in CD2Cl2

AcO

CH2 (Bn)

OBn O

SPh OB n COOMe

67% 1 26b ( C 4)

5 1

3

4

2

6.0

5.8

5.6

5.4

5.2

5.0

4.8

4.6

4.4

4.2

4.0

3.8

3.6

CDHCl2

3.4

3.2

ppm

3.2

ppm

COOMe(b)

B -50°C in CD2Cl2 CH2 (Bn)

1a

6.0

146   

146

5.8

5.6

COOMe(a) 2b

5b

5a

4b

1b

3b 2a

4a

5.4

5.2

5.0

4.8

4.6

4.4

4.2

4.0

3a

3.8

3.6

3.4

Summary, Mechanistic Aspects, and Perspective   

of the observed peak splitting patterns it was concluded that donor 26 predominantly exists in the uncommon 1C4 conformer 26b (67%) along with 33% of the regular 4C1 conformation 26a (Figure 5B).10,11 During warming of the NMR probe to +25°C the peaks of both conformers coalesce resulting in the average spectrum displayed in Figure 5A. In the 1C4 conformer 26b, the electron withdrawing C5 carboxylate and the ring O-atom benefit from their opposite dipole moments (Figure 6). The 4C1 conformation 26a lacks this stabilizing effect. Furthermore, due to the low electronegativity of the sulfur atom (2.44) compared to that of oxygen (3.50), the anomeric effect is less pronounced thereby allowing the C1 thiophenyl group to adopt the equatorial position. Figure 6.

MeOO C

MeOOC OBn O AcO BnO SPh 4

OBn O

MeOO C OB n O A cO BnO

SPh OB n

SPh

MeOO C O Bn O BnO TCAO SP h

A cO 1

C1

C4 favor ed dipoles

26a

26 b

27

28

This conformational equilibrium was also determined in disaccharides having a α-oriented thiophenyl function at their reducing end (Figure 7). An example is disaccharide 32, which was obtained from reaction between hemiacetal donor 30 and acceptor 31.12 Donor 30 was prepared from known 1-thio mannuronic acid ester 2913 via NIS/TFA-mediated hydrolysis.14 After separation of the anomers, which was only possible by reverse-phase high performance liquid chromatography,15 room temperature NMR-analysis of the individual α- and the βanomers of disaccharide 32 revealed that the reducing end thiophenyl uronic acid residue exists as a stable mixture of two conformers. The exact nature of these conformers is not established. Furthermore, the conformational ratio present in the α-disaccharide 32α is different from the ratio found in the β-disaccharide 32β. Exchange of the α-thiophenyl group for an α-glycoside moiety shifts the conformational equilibrium completely towards the 4C1 conformer (cf Chapter 4, compounds 14, 21, and 23). Inspection of the 1H NMR spectra of revealed a small coupling constant between H1 and H2 usually observed in mannopyranoside residues adopting the 4C1 conformation. Figure 7. MeOOC OB n O B nO B nO NIS/TFA DCM/H 2O 6 1%

29 : R = α -SPh 30 : R = α /β-O H

+

MeOOC HO BnO

OBn O

Ph2SO, Tf 2O TTB P, DCM 7 0%

R 31

SPh

MeOO C OBn O BnO BnO MeOO C OBn O O BnO 32 (α / β = 13/87)

SPh

147   

147

Chapter 9   

Having established that donor 26 predominantly exists in the unexpected 1C4 conformer, attention was focused on the exact nature of the reactive intermediate formed after its activation with the Ph2SO/Tf2O reagent combination. In Figure 8 three possible reaction intermediates 33a, 33b and 34 are depicted. Formation of α-triflate intermediate 33b (1C4 conformer) can, a priori, be ruled out due to the unfavorable equatorial positioning of the triflate substituent. Activation of donor 26b (1C4 conformer) would result in initial formation of phenyl sulfonium salt 34. This reactive intermediate 34 is stabilized by the reverse anomeric effect, first described by Lemieux and coworkers.16 In the next step, this phenyl sulfonium substituted intermediate 34 collapses towards the α-triflate 33a. Preliminary low temperature NMR experiments with donor 26 showed, amongst others, formation of two new carbohydrate compounds upon addition of Ph2SO and Tf2O (Figure 9). On the basis of the coupling constants it was concluded that one compound has the 1C4 conformation and the other adopts the 4C1 conformation. The anomeric signal of the 1C4 conformer resonates at δ = 6.51 ppm with a three-bond coupling (3J1,2) of 10.0 Hz (Figure 9A). On the basis of this result, sulfonium species 34 is postulated to be one of the key reaction intermediates (Figure 8). It is assumed that this intermediate 33a is displaced by the acceptor nucleophile in an SN2type fashion resulting in the high to exclusive β-selectivities observed. The other major new reactive intermediate (assigned as 1’, 2’, etc in Figure 9A and B) formed has an anomeric signal appearing as singlet and resonating at δ = 4.34 ppm. After addition of CD3OD to the reaction mixture nothing changed and it was concluded that this compound was the major decomposition product from reaction of donor 26 with Ph2SO and Tf2O. After purification, bicyclic product 36 could be assigned based on NMR- and MS-spectroscopic analysis (Figure 9B).17 This electrophilic aromatic substitution has also been found by Crich and coworkers during activation of 3,4-locked mannopyranosides.18 Apparently, no evidence for triflate formation was found on the basis of these NMR measurements.6b,19 More insight into the glycosylation properties of these deactivated mannuronic acid ester donors can be obtained by using β-thiophenyl derivative 27 (Figure 6).20 The different orientation of the anomeric thiophenyl group possibly leads to different outcomes in the glycosylation of acceptor monosaccharides. Figure 8.

MeOOC OBn O AcO BnO OTf 33a

MeO OC

OB n O

AcO 33b

MeOOC OTf OBn

Ph R O

AcO

OTf S

R

S Ph

34 R = OB n

MeOO C

Ph

OTf O

O O

OTf BnO

O Bn

35

Another point which still has to be addressed is the high α-selectivity in the 3-O-acetyl protected mannosazido donor series. Tricyclic intermediate 35 was postulated as a possible

148   

148

Summary, Mechanistic Aspects, and Perspective   

intermediate leading to full α-selectivity in the coupled disaccharides (Figure 8). Conclusive evidence for the 3-O-acetyl participation model could be gained by application of 3-Otrichloroacetyl protected compound 28 (Figure 6).21 Due to the non-participating character of this group, complete β-selectivity is expected using this donor 28. Figure 9. 1H NMR spectrum of activated donor 26 at -30°C (A) and -10°C (B). A

MeO OC

Ph S

R O

AcO 1

R

S Ph

Ph OTf

1´

5´

34, R = O Bn

2´

4´

1

6.5

6.0

3´

3

5.5

5.0

4.5

4.0

2

3.5

ppm

COOMe´

CDHCl2

B

COOMe´

5

4

J1,2 = 10.0 Hz

7.0

COOMe

CDHCl2

OTf

MeOOC O O AcO BnO

1´ 4´

36

5´ 2´ 3´

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

ppm

References and Notes 1 2

In the absence of a participating group at the C2-position of the donor. (a) Fraser-Reid, B.; Wu, Z.C.; Andrews, W.; Skowronski, E. J. Am. Chem. Soc. 1991, 113, 1434–1435 (b) Andrews, C.W.; Rodebaugh, R.; Fraser-Reid, B. J. Org. Chem. 1996, 61, 5280–5289.

3

The importance of conformationally armed donors was recently underscored by the group of Bols which chemoselectively coupled an armed 1C4 glucosyl donor with an armed 4C1 glucosyl acceptor: Jensen, H.H.; Bols, M. Acc. Chem. Res. 2006, 39, 259–265. It should be mentioned that the exact nature of any transition state during a glycosylation could only be approximated by means of quantum mechanical studies. In addition, a transition state is always

4

5 6

7 8

characterized by the development of substantial oxocarbenium ion character, inevitably leading to conformational alterations. Paulsen, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 155–173. This has already been suggested for 4,6-O-benzylidene protective groups: (a) Fraser-Reid, B.; Wu, Z.; Andrews, C. W.; Skowronski, E.; Bowen, J. P. J. Am. Chem. Soc. 1991, 113, 1434–1435 (b) Crich, D.; Sun, S. J. Am. Chem. Soc. 1997, 119, 11217–11223 (c) Crich, D.; Sun, S. Tetrahedron 1998, 54, 8321–8348 (d) Crich, D. J. Carbohydr. Chem. 2002, 21, 667–690 (e) Crich, D.; Chandrasekera, N.S. Angew. Chem. Int. Ed. 2004, 43, 5386–5389. The Δ2-effect is best known in 4C1-configured mannose donors. (a) Reeves, R.E. J. Am. Chem. Soc. 1950, 72, 1499–1506 (b) Litjens, R.E.J.N. Thesis Leiden University, 2005. In 1940 it was already reported that α-OMe substituted 1C4 2,4-dimethyl-3,6-anhydrogalactopyranose slowly isomerizes to the β-OMe derivative upon standing. This unusual instability was attributed to the Δ2position of the α-OMe anomer. Haworth, W.N.; Jackson, J.; Smith, F. J. Chem. Soc. 1940, 620–632. 149   

149

Chapter 9    9 Spijker, N.M.; Van Boeckel, C.A.A. Angew. Chem., Int. Ed. Engl. 1991, 30, 180–183. 10 Ring proton 1H-NMR data for both conformers: 21a δ = 3.66 (d, J = 9.6 Hz, H-3), 3.98 (s, H-2), 4.56 (d, J = 9.6 Hz, H-5), 5.24 (t, J = 9.6 Hz, H-4), 5.64 (s, H-1); 21b δ = 3.54 (d, J = 12.8 Hz, H-2), 3.78 (s, H-3), 4.47 (s, H-5), 5.51 (s, H-4), 5.74 (d, J = 10.4 Hz, H-1). 11 Such conformational changes to stabilize conflicting electronic interactions present in the monosaccharide ring have more often been described. For example, peresterified β-xylopyranosyl chloride preferred the 1C4 conformation due to the anomeric effect. Lichtenthaler, F.W.; Rönninger, S.; Kreis, U. Liebigs Ann. Chem. 1990, 1001–1006. 12 Garcia, B.A.; Gin, D.Y. J. Am. Chem. Soc. 2000, 122, 4269–4279. 13 See Chapter 4 (compound 7) for method of synthesis. 14 Dinkelaar, J.; Witte, M.D.; Van den Bos, L.J.; Overkleeft, H.S.; Van der Marel, G.A. Carbohydr. Res. 2006, 341, 1723–1729. 15 Separated by RP-HPLC. Column: Gemini C18 5µm, 21x150mm, Eluent: A: 5% ACN/H2O + 0.1% TFA, B:100% ACN, 25 mL/min, gradient: 82%-92% (10 min). 16 (a) Lemieux, R.U.; Morgan, A.R. Can. J. Chem. 1965, 43, 2205–2213 (b) Lemieux, R.U. Pure Appl. Chem. 1971, 25, 527–548. 17 Personal communication J.D.C. Codée, Ph.D. Spectroscopic data for compound 36: 1H NMR (400 MHz, CDCl3) δ = 2.02 (s, 3H, CH3 Ac), 3.69 (s, 3H, CH3 COOMe), 3.76 (dd, 1H, J = 10.0 Hz, J = 3.6 Hz, H-3), 3.91 (d, 1H, J = 3.9 Hz, H-2), 4.05 (d, 1H, J = 9.6 Hz, H-5), 4.24 (s, 1H, H-1), 4.71 (d, 1H, J = 12.4 Hz, CHHPh), 4.72 (d, 1H, J = 15.2 Hz, CHHPh), 4.74 (d, 1H, J = 12.4 Hz, CHHPh), 5.08 (d, 1H, J = 15.2 Hz, CHHPh), 5.51 (t, 1H, J = 10.0 Hz, H-4), 7.04 – 7.37 (m, 14H, CH Arom); 13C NMR (100 MHz, CDCl3) δ = 20.8 (CH3 Ac), 52.7 (CH3 COOMe), 68.2 (CH2 Bn), 68.7 (C-4), 71.9 (CH2 Bn), 72.2 (C-1), 72.8 (C-2), 77.3 (C-5), 77.9 (C-3), 124.2 – 130.6 (CH Arom), 130.7 (Cq Bn, ortho substitution), 135.4 (Cq ipso 2-OBn), 137.8 (Cq 3-OBn), 169.5 (C=O COOMe and Ac). 18 Crich, D.; Cai, W.; Dai, Z. J. Org. Chem. 2000, 65, 1291–1297. 19 The H1 of anomeric triflates usually resonate between δ = 6 – 7 ppm (a) Crich, D.; Cai, W. J. Org. Chem. 1999, 64, 4926–4930 (b) Crich, D.; Smith, M. J. Am. Chem. Soc. 2001, 123, 9015–9020 (c) Wei, P.; Kerns, R.J. J. Org. Chem. 2005, 70, 4195–4198. 20 For the synthesis of β-thiophenyl mannosides see (a) Pedretti, V,; Veyrieres, A.; Sinaÿ, P. Tetrahedron, 1990, 46, 77–88 (b) Crich, D.; Hutton, T.K.; Banerjee, A.; Jayalath, P.; Picione, J. Tetrahedron Asymm. 2005, 16, 105–119. 21 (a) Helferich, B.; Müller, W.M.; Karbach, S. Liebigs Ann. Chem. 1974, 1514–1521 (b) Van Boeckel, C.A.A.; Beetz, T.; Van Aelst, S.F. Tetrahedron 1984, 40, 4097–4107.

150   

150

      Samenvatting          Polysacchariden behoren tot de meest complexe klasse van biopolymeren. Dit complexe karakter is er mede debet aan dat de rol die polysacchariden spelen in biologische systemen niet goed bekend is. Studies naar structuur-activiteits relaties worden sterk gehinderd door een tekort aan zuivere en goed gedefinieerde verbindingen. Een oplossing voor dit probleem is het ontwikkelen van methodieken om fragmenten van polysacchariden (zg. oligosacchariden) te synthetiseren. Het onderzoek dat in dit proefschrift wordt beschreven is gericht op het ontwikkelen en toepassen van (nieuwe) synthese methodieken ter verkrijging van zwitterionische of negatief geladen oligosacchariden. Een gemeenschappelijk kenmerk van deze moleculen is dat ze zogenaamde uronzuren bevatten. Dit zijn suikermoleculen waarvan de C5 hydroxymethyl groep is geoxideerd tot een carbonzuur functie. In Hoofdstuk 1 wordt een (historisch) overzicht gegeven van de verschillende manieren om negatief en zwitterionisch geladen oligosacchariden te bereiden. Er zijn twee benaderingswijzen gangbaar, namelijk 1) het koppelen van alle monosacchariden gevolgd door oxidatie in het stadium van oligosaccharide, of 2) oxidatie van de monosacchariden gevolgd door het koppelen van de uronzuren tot oligosacchariden. De laatste variant vereist zowel een efficiënt oxidatie reagens als een krachtig activator systeem voor het condenseren van de uronzuren. Hoofdstuk 2 beschrijft het gebruik van TEMPO als efficiënt reagens voor de chemo- en regioselectieve oxidatie van 4,6onbeschermde 1-thioglycosiden. Verder wordt aangetoond dat de aldus verkregen inreactive 1-thio uronzuren gebruikt kunnen worden als bouwstenen voor de synthese van een negatief geladen trisaccharide. Recent ontwikkelde sulfonium activator systemen bleken krachtig genoeg te zijn om koppeling van deze inreactieve uronzuren te bewerkstelligen. In Hoofdstuk 3 wordt het gebruik van 3,6-onbeschermde 1-thioglycosiden beschreven. Behandeling van deze verbindingen met het TEMPO reagens gaf de overeenkomstige 6,3-lactonen. Het koppelen van deze tricyclische donor bouwstenen verloopt in het algemeen stereospecifieker dan met de overeenkomstige 1-thio uronzuren beschreven in Hoofdstuk 2. De toename van rigiditeit in de ringstructuur is hier waarschijnlijk de oorzaak van. Verder werd aangetoond

151   

151

 

dat de lactonen efficiënt gebruikt kunnen worden in oligosaccharide synthese. De lacton functie zorgt ervoor dat het aantal beschermende groep manipulaties in het disaccharide stadium tot een minimum beperkt kon worden. Hoofdstuk 4 behandelt de glycosylerende eigenschappen van 1-thiomannuronzuren. De combinatie van het grote anomere effect en de aanwezigheid van een C6 carboxylaat groep maakt dat de koppelingen exclusief β-selectief verlopen. Deze vondst maakt de eerste chemische synthese van een alginaat trisaccharide mogelijk. Tevens werd de invloed van andere elektronenzuigende groepen op de glycosylerende eigenschappen van mannose derivaten onderzocht. Hiertoe werd 1thiomannosazide gefunctionaliseert met verschillende deactiverende groepen. In Hoofdstuk 5 wordt beschreven dat zelfs door de aanwezigheid van 4,6-di-acetyl bescherming al redelijke β-selectiviteit behaald wordt. Toepassing van 3-O-acetyl bescherming resulteerde daarentegen altijd in α-selectiviteit. Hoofdstuk 6 beschrijft een nieuwe synthesemethode voor het verkrijgen van axiaal georiënteerde C4 amino glycosiden. Met behulp van deze een-pots reactie werden vanuit 4,6-onbeschermd gluco- en mannopyranoside op een makkelijke manier 4-amino gesubstitueerde galacto- en talopyranosiden gesynthetiseerd. De laatste twee synthese-hoofdstukken van dit proefschrift beschrijven toepassingen van de ontwikkelde synthesemethodieken. In Hoofdstuk 7 wordt de synthese behandeld van een beschermd trisaccharide dat correspondeert met de repeterende eenheid van het Sp1 zwitterionisch polysaccharide. Verder wordt een nieuwe syntheseroute naar een orthogonaal gefunctionaliseerde 2,4-diamino fucose bouwsteen beschreven. In Hoofdstuk 8 wordt de synthese van een beschermd tetrasaccharide beschreven. Dit is de repeterende eenheid van het zwitterionische polysaccharide PSA1 dat geïsoleerd is uit de celwand van een anaerobe bacterie. Een deel van dit tetrasaccharide werd gesynthetiseerd met behulp van een een-pots dehydratieve condensatiereactie. Het gebruik van deze krachtige synthesetechniek verkleinde het aantal reactiestappen en daarmee het verlies van waardevolle bouwstenen aanmerkelijk. Het bleek echter dat de laatste koppeling niet efficiënt verliep waarschijnlijk vanwege teveel sterische hindering in het acceptor molecuul. Het uitleidende Hoofdstuk 9 geeft naast een korte samenvatting een uitgebreide mechanistische beschrijving van de reactieve intermediairen die gevormd kunnen worden tijdens een koppelingsreactie. De beschreven theorie is daarna getoetst aan de hand van lage temperatuur NMR studies.

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      List of Publications          Sequential one‐pot glycosylations using 1‐hydroxyl and 1‐thiodonors.   J.D.C. Codée, L.J. van den Bos, R.E.J.N. Litjens, H.S. Overkleeft, J.H. van Boom, G.A. van der  Marel Org. Lett. 2003, 5, 1947 – 1950.    A novel strategy towards the synthesis of orthogonally functionalised 4‐aminoglycosides.  L.J.  van  den  Bos,  J.D.C.  Codée,  J.H.  van  Boom,  H.S.  Overkleeft,  G.A.  van  der  Marel  Org.  Biomol. Chem. 2003, 1, 4160 – 4165.    Chemoselective glycosylations using sulfonium triflate activator systems.  J.D.C. Codée, L.J. van den Bos, R.E.J.N. Litjens, H.S. Overkleeft, C.A.A. van Boeckel, J.H. van  Boom, G.A. van der Marel Tetrahedron 2004, 60, 1057 – 1064.     Thioglycuronides: Synthesis and application in the assembly of acidic oligosaccharides.   L.J. van den Bos, J.D.C. Codée, J.C. van der Toorn, T.J. Boltje, J.H. van Boom, H.S. Overkleeft,  G.A. van der Marel Org. Lett. 2004, 6, 2165 – 2168.     Sulfonium triflate mediated glycosidations of aryl 2‐azido‐2‐deoxy‐1‐thio‐D‐mannosides.  R.E.J.N.  Litjens,  L.J.  van  den  Bos,  J.D.C.  Codée,  R.J.B.H.N.  van  den  Berg,  H.S.  Overkleeft,  G.A. van der Marel Eur. J. Org. Chem. 2005, 5, 918 – 924.    Preparation of 1‐thio uronic acid lactones and their use in oligosaccharide synthesis.  L.J.  van  den  Bos,  R.E.J.N.  Litjens,  R.J.B.H.N.  van  den  Berg,  H.S.  Overkleeft,  G.A.  van  der  Marel Org. Lett. 2005, 7, 2007 – 2010.    Thioglycosides in sequential glycosylation strategies.  J.D.C.  Codée,  R.E.J.N.  Litjens,  L.J.  van  den  Bos,  H.S.  Overkleeft,  G.A.  van  der  Marel  Chem.  Soc. Rev. 2005, 34, 769 – 782.         

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          Synthesis  of  an  α‐Gal  epitope  α‐D‐Galp‐(1→3)‐β‐D‐Galp‐(1→4)‐β‐D‐GlcNAc‐lipid  conjugate.  R.E.J.N. Litjens, P. Hoogerhout, D.V. Filippov, J.D.C. Codée, L.J. van den Bos, R.J.B.H.N. van  den Berg, H.S. Overkleeft, G.A. van der Marel, J. Carbohydr. Chem. 2005, 24, 755 – 769.    NIS/TFA: a general method for hydrolyzing thioglycosides.  J.  Dinkelaar,  M.D.  Witte,  L.J.  van  den  Bos,  H.S.  Overkleeft,  G.A.  van  der  Marel,  Carbohydr.  Res. 2006, 341, 1723 – 1729.    Stereocontrolled  Synthesis  of  β‐D‐Mannuronic  Acid  Esters:  Synthesis  of  an  Alginate  Trisaccharide.  L.J. van den Bos, J. Dinkelaar, H.S. Overkleeft, G.A. van der Marel J. Am. Chem. Soc. 2006, 128,  13066 – 13067.    The use of bifunctional protective groups in oligosaccharide synthesis – an overview  R.E.J.N.  Litjens,  L.J.  van  den  Bos,  J.D.C.  Codée,  H.S.  Overkleeft,  G.A.  van  der  Marel  Carbohydr. Res. 2007, 342, 419 – 429.    Sequential Glycosylations; a focus on thioglycosides.  G.A. van der Marel, L.J. van den Bos, H.S. Overkleeft, R.E.J.N. Litjens, J.D.C. Codée Frontiers  in  Carbohydrate  Chemistry,  Ed:  A.V.  Demchenko,  ACS  Symposium  Series  No.  960,  Oxford  University Press, New York, 2007, Chapter 12.    Studies  on  the  Glycosidation  Properties  of  1‐Thio‐Mannosazidopyranosides  and  1‐Thio‐ Mannosaziduronic Acid Esters.  L.J. van den Bos, B.A. Duivenvoorden, M.C. de Koning, D.V. Filippov, H.S. Overkleeft, G.A.  van der Marel Eur. J. Org. Chem. 2007, 116 – 124.    A synthetic study toward the PSA1 tetrasaccharide repeating unit.  L.J. van den Bos, T.J. Boltje, T. Provoost, J. Mazurek, H.S. Overkleeft, G.A. van der Marel  Tetrahedron Lett. 2007, in press. 

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      Curriculum Vitae          Leendert Johannes van den Bos werd op 1 maart 1979 geboren in Bruinisse (Zld.). Na het  behalen van het HAVO‐diploma aan het Calvijn College te Goes in 1996 werd in dat jaar een  aanvang  gemaakt  met  de  studie  Organische  Chemie  aan  de  Hogeschool  Rotterdam  en  Omstreken  te  Delft. In  het  kader  van  het  afstudeeronderzoek  werd  van september  1999 tot  juni  2000  onderzoek  verricht  bij  Byk  Gulden  (het  latere  Altana  Pharma  AG)  te  Konstanz  (Dld).  Dit  onderzoek  behelsde  de  synthese  van  heterocyclische  aromaten  als  mogelijke  tryptase  inhibitoren  die  van  belang  kunnen  zijn  voor  de  behandeling  van  astma.  De  opleiding  werd  in  2000  met  goed  gevolg  afgerond  en  in  september  van  dat  jaar  werd  een  aanvang  gemaakt  met  de  studie  Scheikunde  aan  de  Universiteit  Leiden.  In  de  periode  van  september 2001 tot en met mei 2002 werd de hoofdvakstage uitgevoerd bij de vakgroep Bio‐ organische  Synthese  onder  leiding  van  prof.  dr.  J.H.  van  Boom  en  prof.  dr.  G.A.  van  der  Marel.  De  stage  was  gericht  op  de  synthese  van  het  α‐Gal  trisaccharide  voorzien  van  een  vetstaart;  dit  als  onderdeel  van  een  immunologische  studie  naar  een  mogelijk  vaccin  tegen  kanker. Het doctoraal examen werd in 2002 behaald.  Van  september  2002  tot  september  2006  werd  als  assistent‐in‐opleiding  het  in  dit  proefschrift  beschreven  onderzoek  uitgevoerd  bij  de  vakgroep  Bio‐organische  Synthese  onder leiding van prof. dr. G.A. van der Marel en prof. dr. H.S. Overkleeft. Delen van de in  dit proefschrift beschreven resultaten zijn gepresenteerd op de jaarlijkse bijeenkomst van de  Holland Research School of Molecular  Chemistry te Amsterdam (Januari  2003,  mondelinge  presentatie),  het  22nd  International  Carbohydrate  Symposium  te  Glasgow,  Schotland  (Juli  2004, poster presentatie), het CEDNETS Symposium te Krakau, Polen (Juni 2005, mondelinge  presentatie)  en  het  23rd  International  Carbohydrate  Symposium  te  Whistler  (BC),  Canada  (Juli 2006, poster presentatie).  Sinds  november  2006  wordt  in  samenwerking  met  het  biotechnologiebedrijf  Crucell  onderzoek  verricht  naar  de  synthese  van  suikerverbindingen  van  bacteriële  oorsprong  en  bijbehorende biologische activiteit.    155   

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      Nawoord          De  laatste  pagina  van  dit  proefschrift  wil  ik  graag  gebruiken  voor  het  bedanken  van  de  vele  personen  die  op  enigerlei  wijze  betrokken  zijn  geweest  bij  mijn  studie  en  promotie.  Hierbij wil ik beginnen met mijn ouders die mij altijd gestimuleerd hebben om door te leren  (“je  moet  het  nu  doen;  als  je  ouder  wordt  gaat  het  niet  meer”).  Verder  natuurlijk  mijn  schoonmoeder, (schoon)broers en (schoon)zussen voor het plezier en de steun die jullie mij  gegeven hebben toen ik studeerde en mijn promotieonderzoek uitvoerde.   Een  niet  onbelangrijk  deel  van  het  hier  beschreven  onderzoek  is  uitgevoerd  door  studenten. Achtereenvolgens zijn dat Tomasz, Thomas, Boudewijn, Tom en Anwar geweest.  Ik heb veel van jullie geleerd en had er altijd veel plezier in om met jullie samen te werken.  Zahra, I appreciated your short stay in our lab. Voor jullie allemaal geldt, bedankt voor alles  en veel succes met jullie toekomstplannen.   Alle collega’s van de vakgroep Bio‐organische Synthese wil ik ook graag bedanken.  Zowel  de “ouderen” als de lichting nieuwe aio’s zorgden ervoor dat er altijd een prettige werksfeer  was. Een aparte vermelding verdienen mijn mede “sugarboys” Jeroen en Remy. Ik heb altijd  erg  prettig  met  jullie  samengewerkt  en  vele  mooie  ideeën  (al  dan  niet  succesvol)  uitgeprobeerd. Ook Richard en Martijn de K. wil ik graag bedanken voor de gezelligheid en  wetenschappelijke  deliberaties.  Evenals  Erwin  waar  ik  al  10  jaar  dezelfde  chemische  interesses mee deel. Verder heb ik de contacten met Bas, Steven, Paul van S., Micha, Michiel,  Tom,  Jimmy,  Jasper,  Gijsbert,  Mattie,  Kimberly,  Farid,  Karen,  Martijn  V.,  Peter,  Carlo,  Henrik,  Ali,  Vicky,  Paul  G.,  Varsha,  Martijn  R.,  Matthijs,  Rian,  Martin,  Christoph  en  Ulrik  altijd  zeer  prettig  en  gezellig  gevonden.  De  hulp  van  Hans  en  Nico  voor  het  meten  van  (LC)MS en het zuiveren van oligosacchariden was onmisbaar. Van de NMR afdeling wil ik  Kees en Fons hartelijk bedanken voor de hulp bij het maken van strakke platen. Verder ook  de ama’s; bedankt voor de technische ondersteuning.  Lies,  ontzettend  bedankt  voor  je  liefde  en  steun  de  afgelopen  jaren.  Je  bent  met  me  meegegaan uit dat mooie Zeeland naar Moordrecht. Ik hoop op nog vele jaren samen met jou  en de kindjes.  157   

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